Alkaline proteases and detergents and cleaners comprising these alkaline proteases

ABSTRACT

The present application relates to two novel proteases (SEQ ID NO: 4 and 7) which are similar to one another. The DNA of the two proteases, was obtained from soil samples, and C-terminally deleted, producing proteolytically active fragments thereof (SEQ ID NO: 5 and 8). The invention also provides all alkaline proteases similar at least to 90% to SEQ ID NO: 4 or to 87.5% to SEQ ID NO: 7, and those which can be summarized under a consensus sequence (SEQ ID NO: 9) derived from SEQ ID NO: 4 and 7. Furthermore, it relates to all nucleic acids which have a homology of at least 85% identity to the associated nucleic acids (SEQ ID NO: 3 and 6) or the fragments concerned. Furthermore, it defines technical possibilities of use for these proteases and especially describes their use in detergents and cleaners.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/552,061filed 23 Oct. 2006, now issued U.S. Pat. No. 7,691,618, which is acontinuation of PCT/EP2005/003983 filed 15 Apr. 2005, which in turnclaims priority to German Patent Application No. 10 2004 019 751.2 filed23 Apr. 2004, each of which is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 22, 2011, isnamed 20131002.txt and is 58,059 bytes in size.

FIELD OF THE INVENTION

The present application relates to two novel alkaline proteases whichare similar to one another, encoded by DNA obtained from soil samples,to C-terminally deleted, likewise proteolytically active fragmentsthereof and all sufficiently similar alkaline proteases and nucleicacids, and methods of use of these proteases, particularly in detergentsand cleaners.

BACKGROUND OF THE INVENTION

Proteases belong to the industrially most important enzymes in general.Among these, in turn, serine proteases of the subtilisin type(subtilases, subtilopeptidases, EC 3.4.21.62) which containcatalytically active amino acids are particularly important. Theseenzymes are nonspecific endopeptidases, i.e., they hydrolyze acid amidebonds which lie in the interior of peptides or proteins. Their pHoptimum usually lies in the distinctly alkaline range. The article“Subtilases: Subtilisin-like Proteases” by R. Siezen, SubtilisinEnzymes, ed. R. Bott and C. Betzel, (New York 1996), pp. 75-95, forexample, offers an overview of this family. Subtilases are naturallyformed from microorganisms. Among these subtilases, the subtilisinsformed and secreted by Bacillus species are of particular interest.

Proteases are established, active ingredients present in a variety ofdetergents and cleaners as they catalyze the breakdown ofprotein-containing soil on the goods to be cleaned. Ideally, synergisticeffects result between the enzymes and the other constituents present inthe compositions concerned. Among the detergent and cleaner proteases,subtilases are particularly preferred due to their favorable enzymaticproperties, including stability and pH optimum. In addition, they arealso suitable for a large number of further industrial uses, forexample, as constituents of cosmetics and in the synthesis of organicchemicals.

Microorganism-containing samples from natural habitats may be culturedunder conditions suitable for the production of novel enzymes, e.g.,under alkaline conditions. In this way, novel alkaline proteases may beisolated. The microorganisms producing the most efficient enzymes arethen selected for and purified, for example by means of plating out onprotein-containing agar plates and measuring the lysis halos formed.Optionally, the genes encoding the proteases may be cloned. Such aprocedure is described, for example, in the textbook AlkalophilicMicroorganisms. A new microbial world, by K. Horikoshi and T. Akiba(Japan Scientific Societies Press, Springer-Verlag, N.Y., Heidelberg,Berlin, ISBN 0-387-10924-2, 1982), Chpt. 2, pp. 9-26.

Notably, microbially-produced alkaline proteases are already employed indetergents and cleaners. For example, see WO 93/07276 A1 which describesthe protease 164-A1 from Chemgen Corp., Gaithersburg, Md., USA, andVista Chemical Company, Austin, Tex., USA, obtainable from Bacillusspec. 164-A1 is suitable for use in detergents and cleaners. Otherexamples are the alkaline protease from Bacillus sp. PD138, NCIMB 40338from Novozymes A/S, Bagsvaerd, Denmark (WO 93/18140 A1), the proteinaseK-16 from Kao Corp., Tokyo, Japan (U.S. Pat. No. 5,344,770), originatingfrom Bacillus sp. ferm. BP-3376 and the protease described in WO96/25489 A1 (Procter & Gamble, Cincinnati, Ohio, USA) from thepsychrophilic organism Flavobacterium balustinum.

Natural proteases may be optimized, for use in detergents and cleaners,via mutagenesis methods known in the art. Such methods include pointmutagenesis, (e.g., generation of deletion, or insertion mutants) orfusion with other proteins or protein parts. The strategy of introducingspecific point mutations into the known subtilisin molecules, in orderto improve the washing performance, is also referred to as rationalprotein design. A similar strategy to improve performance entailsmodifying the surface charges and/or the isoelectric point of themolecules in order to modulate their interactions with the substrate viathe introduction of point mutations. A further, supplementary strategyconsists of increasing the stability of the proteases thereby increasingtheir efficacy. Stabilization by means of coupling to a polymer isdescribed for proteases used in cosmetics, for example, in U.S. Pat. No.5,230,891. Such proteases exhibit improved skin compatibility. However,for detergents and cleaners, stabilization by point mutations is morecommonly employed.

A new approach in enzyme development entails combining elements ofrelated, known proteins thereby generating novel enzymes having improvedfunctional properties. Such methods are also referred to as “directedevolution”. These include, without limitation: The StEP method (Zhao etal., Nat. Biotechnol., Vol. 16 (1998), pp. 258-261), Random primingrecombination (Shao et al., Nucleic Acids Res., Vol. 26 (1998), pp.681-683), DNA shuffling (Stemmer, W. P. C., Nature, Vol. 370 (1994), pp.389-391) or RACHITT (Coco, W. M. et al., Nat. Biotechnol., Vol. 19(2001), pp. 354-359). A further shuffling method referred to as“Recombining ligation reaction” (RLR) is described in WO 00/09679 A1.

Below, an overview of the industrially most important alkaline proteasesof the subtilisin type is provided. Subtilisin BPN′, which originatesfrom Bacillus amylotiquefaciens, or B. subtilis, is described byVasantha et al. in J. Bacteriol., Vol. 159 (1984), pp. 811-819 and J. A.Wells et al. in Nucleic Acids Research, Vol. 11 (1983), pp. 7911-7925.Subtilisin BPN′ is used as a reference enzyme with respect to numberingof amino acid positions in the subtilisins.

For example, the position of point mutations in subtilisin described inEP 251446 A1, are indicated using the numbering of BPN' as a reference.Procter & Gamble Corp., of Cincinnati, Ohio, USA, refer to this materialas “Protease B”. The BPN' variants of EP 199404 A1 are referred to byProcter & Gamble Corp. as “Protease A”. A “Protease C” is in turncharacterized, according to WO 91/06637 A1, by further point mutationsof BPN'. “Protease D” refers, according to WO 95/10591 A1, to variantsof the protease from Bacillus lentus.

The protease subtilisin Carlsberg is described in the publications of E.L. Smith et al. in J. Biol. Chem., Vol. 243 (1968), pp. 2184-2191, andJacobs et al. in Nucl. Acids Res., Vol. 13 (1985), pp. 8913-8926. It isformed naturally by Bacillus licheniformis, and was and is obtainableunder the trade name Maxatase® from Genencor International Inc.,Rochester, N.Y., USA, and under the trade name Alcalase® from NovozymesA/S, Bagsvaerd, Denmark.

Protease PB92 is produced naturally by the alkalophilic bacteriumBacillus nov. spec. 92 and is obtainable under the trade name Maxacal®from the Gist-Brocades company, Delft, Netherlands. It is described inits original sequence in Patent Application EP 283075 A2.

Subtilisins 147 and 309 are marketed under the trade names Esperase® andSavinase®, respectively, by Novozymes. They were originally obtainedfrom Bacillus strains that are disclosed by Application GB 1243784 A.

Subtilisin DY was originally described by Nedkov et al. in Biol. Chem.Hoppe-Seyler, Vol. 366 (1985), pp. 421-430.

The alkaline protease from B. lentus is an alkaline protease fromBacillus species and is described in Application WO 91/02792 A1. Itnatively possesses comparatively good stability with respect tooxidation and the action of detergents. Application WO 91/02792 A1 andPatents EP 493398 B1 and U.S. Pat. No. 5,352,604 describe itsheterologous expression in the host B. licheniformis ATCC 53926. Theclaims of the aforesaid US patent refer to positions 208, 210, 212, 213,and 268 as characteristic of the B. lentus alkaline protease; theycorrespond, in the numbering of the mature protein, to positions 97, 99,101, 102, and 157. However this enzyme differs from the maturesubtilisin 309 (Savinase®). The three-dimensional structure of thisenzyme is described in “The crystal structure of the Bacillus lentusalkaline protease, Subtilisin BL, at 1.4 Å resolution”, Goddette et al.,J. Mol. Biol., Vol. 228 (1992), pp. 580-595. Industrially importantvariants of this enzyme that are stabilized by point mutagenesis and aresuitable in particular for use in washing and cleaning products aredisclosed, inter alia, in Applications WO 92/21760 A1, WO 95/23221 A1,WO 02/088340 A2, and WO 03/038082 A2.

The enzyme thermitase, formed naturally by Thermoactinomyces vulgaris,was originally described by Meloun et al. (FEBS Lett. (1983), pp.195-200). This is a molecule that as a whole exhibits substantialsequence discrepancies compared with the other subtilisins. The homologybetween the mature thermitase and the alkaline protease proteins from B.lentus DSM 5483 (see below) is not very high, (e.g., 45% identity; 62%similar amino acids).

Proteinase K is also a protease that exhibits comparatively low homologywith the alkaline protease from B. lentus: only 33% identity (46%similar amino acids) at the level of the mature proteins. Proteinase Kderives originally from the microorganism Tritirachium album Limber, andhas been described by K.-D. Jany and B. Mayer in Biol. Chem.Hoppe-Seyler, Vol. 366 (1985), pp. 485-492.

WO 88/07581 A1 discloses proteases TW3 and TW7, which are very similarto one another, for use inter alia in washing and cleaning products.

Bacillopeptidase F from Bacillus subtilis possesses only 30% identity tothe B. lentus alkaline protease at the amino-acid level. This enzyme isdiscussed in the aforementioned work by Siezen et al., but has nothitherto been described or claimed for use in washing and cleaningproducts.

Application WO 01/68821 A2 describes new subtilisins having goodperformance with respect to egg stains.

Further alkaline proteases that are formed from microorganisms that canbe isolated from natural habitats are described in Applications WO03/054185 A1 (from Bacillus gibsonii (DSM 14391)), WO 03/056017 A2 (fromBacillus sp. (DSM 14390)), WO 03/055974 A2 (from Bacillus sp. (DSM14392)), and WO 03/054184 A1 (from Bacillus gibsonii (DSM 14393)). Allthese Applications also disclose corresponding washing and cleaningproducts containing these novel alkaline proteases.

A further group of industrially important proteases are themetalloproteases, e.g., enzymes that require a metal cation as acofactor. Representatives of these are also assigned to the family ofsubtilases. For instance, metalloproteases from gram-positivemicroorganisms such as B. subtilis, but also from S. cerevisiae, S.pombe, E. coli and H. influenzae, are described in US 2003/0113895 A1.WO 00/60042 A1 and WO 02136727 A1, disclose detergents and cleanerscontaining metalloproteases. DE 10360805.2 discloses an alkalinemetalloprotease whose encoding DNA is obtainable from a soil sample, andits use in detergents and cleaners.

A large number of novel proteases are described in WO 20041033668 A2.StmPr2 from Stenotrophomonas maltophilia, which is deposited under theentry AY253983 in GenBank (National Center for Biotechnology InformationNCBI, National Institutes of Health, Bethesda, Md., USA), has also beenpreviously described.

Further known protease enzymes are obtainable under the trade namesDurazym®, Relase®, Everlase®, Nafizym, Natalase® and Kannase® fromNovozymes, under the trade names Maxapem®, Purafect®, Purafect OxP® andProperase® from Genencor, under the trade name Protosol® from AdvancedBiochemicals Ltd., Thane, India and under the trade name Wuxi® from WuxiSnyder Bioproducts Ltd., China.

In light of the foregoing, it is clear that there is a great need forindustrially employable proteases which exhibit altered activities frompreviously known proteases, particularly for their use in detergents andcleaners. A suitable protease for detergents or cleaners should exhibita certain insensitivity to conditions suitable for cleaning—e.g., thepresence of surfactants which are denaturing, of bleach, and hightemperatures, etc.—and also exhibit catalytic activity againstappropriate substrates such as the proteins found in food residues.

There also exists a need for new alkaline proteases, which are naturallyobtainable but which are also amenable to further optimization by meansof various mutagenesis strategies. Such novel proteases may be generatedusing recently established shuffling technologies. Nucleotide sequences(even if the encoded enzyme exhibits comparatively modest performance)can be shuffled to produce new variants and thus in turn provideentirely new artificial enzymes for use in a variety of industrialapplications.

SUMMARY OF THE INFORMATION

In accordance with the present invention, alkaline proteases areprovided which naturally bring about an improvement in the performanceof washing or cleaning products.

Also included in the scope of the invention are methods for isolatingsuch proteases. Further objects include provision of nucleic acids thatencode such proteases and isolated proteases produced by expression ofthe same. Yet another object entails genetic-engineering such nucleicacids to develop improved proteases (e.g., by a shuffling procedure).The proteases obtained by the expression of the engineered nucleic acidsalso comprise an aspect of the invention. Such proteases may be used toadvantage in washing and cleaning products and corresponding washing andcleaning methods. The proteases described herein may also be employed inindustrial applications.

The present discovery was not based on conventional enrichment culturefor alkaphilic, protease-producing microorganisms, rather, nucleic acidscoding for alkaline proteases were isolated from soil samples. Becausethe isolated nucleic acids cannot be assigned to a specific strain ofbacteria, such nucleic acids are referred to as “metagenomic DNA”.

Surprisingly, according to this method two novel proteases wereidentified which have an extraordinary similarity to one another and areemployable successfully in detergents and cleaners as complete matureenzymes or as C-terminal deletion mutants.

Thus the present invention provides an alkaline protease comprising anamino acid sequence which is identical to the amino acid sequence of SEQID NO. 4, or a sequence which is at least 90% identical thereto. Alsoprovided is an alkaline protease having the amino acid sequence of SEQID NO. 7 and a sequence which is at least to 87.5% identical thereto.

Also in accordance with the present invention are nucleic acids encodingthe proteases described above, cells comprising such nucleic acids andmethods for their characterization. Also provided are detergents andcleaners comprising the proteases and methods of use for the same.

As the working examples confirm, enzymes encoded by SEQ ID NO. 4 and 7and their associated mature enzymes exhibit proteolytic activities whichare suitable for use in detergents and cleaners. Provision of the DNAencoding these enzymes facilitates additional optimization of theseenzymes, via introduction of point mutations. Furthermore, these DNAscan be subjected to shuffling procedures, thereby producing completelynovel proteases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Alignment of the alkaline proteases HP70 and HP53 according tothe invention (SEQ ID NO. 4 and 7) with alkaline proteases from theprior art. HP70: alkaline protease shown in SEQ ID NO. 4; HP53: alkalineprotease as shown in SEQ ID NO. 7; SP: extracellular serine protease(SEQ ID NO: 14) (E.C. 3.4.21.-) from Xanthomonas campestris pv.campestris (ATCC 33913) (Accession No. NP_(—)636242 at GenBank); BLAP:alkaline protease (SEQ ID NO: 15) from Bacillus lentus DSM 5483 (WO92/21760 A1).

FIG. 2: Alignment of the genes of the alkaline proteases HP70(nucleotides 1-1746 of SEQ ID NO: 3) and HP53 (SEQ ID NO: 6) accordingto the invention (SEQ ID NO. 3 and 6) with those of alkaline proteasesfrom the prior art. HP70: gene of the alkaline protease HP70 shown inSEQ ID NO. 3; HP53: gene of the alkaline protease HP53 shown in SEQ IDNO. 6; SP: gene of the extracellular serine protease (SEQ ID NO: 16)(E.C. 3.4.21.-) from Xanthomonas campestris pv. campestris (ATCC 33913)(Accession No. NP_(—)636242 at GenBank); BLAP: gene of the alkalineprotease (SEQ ID NO: 17) from Bacillus lentus DSM 5483 (WO 92/21760 A1).

FIG. 3: Schematic representation of the plasmid vector pUC18 used forthe setting up of an expression gene bank as in Example 2. The vectorwas linearized with Sma I for the setting up of the metagenomic DNAdigested with Alu I.: ORI: replication origin Plac: lac promoter;lacZ-alpha: gene for the alpha-peptide of the beta-galactosidase;amp^(R): ampicillin resistance-mediating beta-lactamase.

FIG. 4: Alignment of the amino acid sequences of the two proteases HP70(SEQ ID NO. 4) and HP53 (SEQ ID NO. 7) according to the invention forthe development of the consensus sequence of SEQ ID NO. 9. The aminoacid positions designated therein as variables X can be attributedeither to HP70 or to HP53 as shown in this figure.

DETAILED DESCRIPTION OF THE INVENTION

A protein within the meaning of the present application refers to apolymer composed of linearly synthesized natural amino acids, whichtypically assume a three-dimensional structure for effecting proteinfunction. In the present application, the 19 proteinogenous, naturallyoccurring L-amino acids are designated by the customary 1- and 3-lettercodes. The combination of one of these designations with a numberindicates the particular amino acid residue within the respectiveposition. Analogous designations are established for point mutations.Positional data relate, if not stated otherwise, to the mature forms ofthe proteins, e.g., proteins which lack signal peptides (see below).

An enzyme within the meaning of the present application is to beunderstood as meaning a protein which carries out a certain biochemicalfunction. Proteolytic enzymes or enzymes having a proteolytic functionare to be understood as meaning those which hydrolyze the acid amidebonds of proteins.

Numerous proteins are formed as “preproteins”, that is, they alsoinclude a signal peptide sequence at the N-terminal part of the protein.Signal peptide sequences facilitate expulsion of the protein formed inthe producing cell into the periplasma or the surrounding medium and/orits correct folding. Subsequently, the signal peptide is removed fromthe remainder of the protein under natural conditions by a signalpeptidase, the mature protein exhibiting proteolytic activity.

For industrial applications, mature peptides, that is, enzymes processedas described above, are preferred compared to the pre-proteins.

Pro-proteins are inactive precursors of proteins. Their precursorshaving a signal sequence are designated as pre-pro-proteins.

Nucleic acids serve as information carriers which encode for the linearamino acid sequence in proteins or enzymes. They can be present assingle stranded or as double stranded molecules. As DNA is more stablethan other types of nucleic acids, it is preferred for molecularbiological studies. In another aspect, nucleic acids isolated from thenatural environment (e.g., an RNA) are also within the scope of theinvention. (c)DNA molecules reversed transcribed from such RNA moleculesalso comprise as aspect of the invention.

Genes comprise the information unit of nucleic acids encoding aparticular protein. In DNA, the sequences of both complementary strandsare to be taken into consideration in all three possible reading framesin each case. Furthermore, various codon triplets can code for the sameamino acids, so that certain amino acid sequences can be derived from anumber of different nucleotide sequences, some which exhibit only lowidentity. This phenomenon is referred to as the degeneracy of thegenetic code. Moreover, various organisms show differences in the use ofthese codons. For these reasons, the nucleic acids provided herein areexemplary as variations of these sequences are also within the scope ofthe invention due to the aforementioned degeneracy of the genetic code.

Methods such as chemical synthesis and polymerase chain reaction (PCR)in combination with other conventional molecular biology and/or proteinchemistry methods, facilitate the preparation of complete genes havingknown DNA and/or amino acid sequences. Such methods are described, forexample, in “Lexikon der Biochemie” [Encyclopedia of Biochemistry],Spektrum Akademischer Verlag, Berlin, Vol. 1 (1999), pp. 267-271 andVol. 2, pp. 227-229. For example, known DNA sequences can be amplifiedwith sequence specific PCR primers using isolated mRNA molecules.Nucleic acids from such strains can then be synthesized, cloned and, ifdesired, further mutagenized.

Modifications to the nucleotide sequence are referred to as mutations.Such mutations can include modification, deletion, insertion orsubstitution mutations or those in which various genes or parts of genesare shuffled or fused with one another. The organisms producing suchmodified nucleic acids are designated as mutants. The proteins derivedfrom mutated nucleic acids are designated as variants. For instance,deletion, insertion, substitution mutations or fusions lead todeletion-, insertion-, substitution-mutated or fusion genes and, at theprotein level, to corresponding deletion, insertion or substitutionvariants, or fusion proteins.

Point mutations refer single amino acid replacements and the followingconvention is used first, the naturally present amino acid is designatedin the form of the internationally customary single-letter code,followed by the associated sequence position and finally the insertedamino acid. A number of exchanges within the same polypeptide chain areseparated from one another by obliques.

Vectors within the meaning of the present invention refer to nucleicacids, which contain a gene of interest. Vectors are useful forexpressing the gene of interest in a species or a cell line over anumber of generations or cell divisions as a stable genetic elementreplicating independently of the other genome. Vectors are specialplasmids, that is, circular genetic elements, when used in bacteria. Adifferentiation is made in genetic engineering between those vectorswhich are used for storage, e.g., “cloning vectors”, and those whichfulfill the function of producing the gene of interest in the host cell,e.g., expression vectors.

Both bacterial cells and eukaryotic cells which contain said vectors aregenerally designated as cells regardless of their differences. Thosecells which contain a vector, in particular an expression vector, andcan thus be used for the expression of a transgene, are designated ashost cells, since they accommodate the genetic system concerned.

Homologization is the comparison of a nucleic acid or amino acidsequence with that of known genes or proteins. It is performed, forexample, by means of alignment. The measure of the homology is apercentage of identity, as can be determined, according to the methodindicated by D. J. Lipman and W. R. Pearson in Science, Vol. 227 (1985),pp. 1435-1441. Preferably, it is carried out by means of algorithms,which are provided by commercially obtainable computer programs. Theseinclude, for example, the program Vector NTI® Suite 7.0, obtainable fromInforMax, Inc., Bethesda, USA, preferably using the specified defaultparameters. The homology indication can relate to the entire protein orto the range to be assigned in each case. A more widely used homologyterm, “similarity”, relates to conserved variations, that is, aminoacids having a similar chemical structures which usually exert similarchemical activities within the protein. With nucleic acids, only thepercentage of identity is known.

By homologization, the functions of individual sequence ranges and theenzymatic activity of the entire enzyme can be determined from the aminoacid or nucleotide sequence. Homologous ranges of different proteins arethose having comparable functions, which can be recognized by identityor conserved exchanges in the primary amino acid sequence. They compriseindividual amino acids, very small regions, “boxes”, which are a fewamino acids in length, up to long regions in the primary amino acidsequence. The functions of the homologous regions are thus also to beunderstood as meaning very small subfunctions of the function exerted bythe entire protein, such as, for example, the formation of individualhydrogen bonds for the complexation of a substrate or transitioncomplex. Other regions of the protein which are not involved in theactual enzymatic reaction can qualitatively or quantitatively modifythem. This relates, for example, to the enzyme stability, the activity,the reaction conditions or the substrate specificity.

The term “proteolytic enzyme” or protease is therefore to be understoodas meaning, beyond the functions of the few amino acid residues of thecatalytically active center, all functions such as result due to theaction of the entire other protein or of a part or a number of parts ofthe other protein on the actually catalytically active regions. It ismoreover possible that also the activities of other proteases arequalitatively or quantitatively modified by one or more parts, forexample of the protein according to the invention. This influencing ofother factors is likewise regarded as a proteolytic activity.Proteolytically active enzymes are also those proteases whose activityat a given point in time is blocked, for example by an inhibitor. Theirprincipal suitability is decisive for the corresponding proteolysisreaction.

Fragments are understood as meaning all proteins or peptides which aresmaller than natural proteins or those which correspond to completelytranslated genes, and, for example, can also be obtained synthetically.On account of their amino acid sequences, they can be assigned to thecomplete proteins concerned. They can, for example, assume identicalstructures or exert proteolytic activities or subactivities. Fragmentsand deletion variants of starting proteins are in principle similar;while fragments are more likely smaller pieces, the deletion mutantsmore likely lack only short regions, and thus only individualsubfunctions.

Chimeric or hybrid proteins are to be understood within the meaning ofthe present application as those proteins which are composed of elementswhich naturally originate from different polypeptide chains of the sameorganism or from different organisms. This procedure is also calledshuffling or fusion mutagenesis. The point of such a fusion consists,for example, in bringing about or modifying an enzymatic function withthe aid of the fused-on protein part according to the invention.

Proteins obtained by insertion mutation are to be understood as meaningthose variants which have been obtained by means of methods known per seby insertion of a nucleic acid fragment, or protein fragment into thestarting sequences. They are to be designated as chimeric proteinsbecause of their similarity in principle. They differ from these only inthe size ratio of the unmodified protein part to the size of the entireprotein. In such insertion-mutated proteins, the proportion of foreignprotein is lower than in chimeric proteins.

Inversion mutagenesis, that is a partial sequence reversal, can beregarded as a special form both of deletion, but also of insertion. Thesame applies for a new grouping of different molecular parts differingfrom the original amino acid sequence. It can be regarded as a deletionvariant, as an insertion variant, and as a shuffling variant of theoriginal protein.

Derivatives are understood within the meaning of the present applicationas meaning those proteins whose pure amino acid chain has beenchemically modified. Such derivatizations can be carried out, forexample, biologically in connection with the protein biosynthesis by thehost organism. For this, for example, molecular biological methods, forexample cotransformation with genes which provide for the modificationconcerned, can be employed. Derivatizations, however, can also becarried out chemically, for example by the chemical conversion of a sidechain of an amino acid or by covalent bonding of another compound to theprotein. Such a compound, for example, can also be other proteins which,for example, are bonded to proteins according to the invention by meansof bifunctional chemical bonds. Such modifications, for example,influence the substrate specificity or the binding strength to thesubstrate or bring about a temporary blockage of the enzymatic activity,if the coupled substance is an inhibitor. This is useful, for example,for the storage time period. Likewise, derivatization is to beunderstood as meaning covalent bonding to a macromolecular carrier.

The performance of an enzyme is understood as meaning its activity inthe industrial field, preferably in the context of a correspondinglyaligned composition. This is based on the actual enzymatic activity, butmoreover depends on further factors relevant to the particular process.These include, for example, stability, substrate binding, interactionwith the material carrying the substrate or interactions with otherconstituents, in particular synergies.

The washing performance or the cleaning performance of a detergent, orcleaner, is to be understood within the meaning of the presentapplication as meaning the effect which the composition consideredexerts on the soiled article, for example textiles or articles with hardsurfaces. Individual components of such compositions, for exampleindividual enzymes, are assessed with respect to their contribution tothe washing or cleaning performance of the entire detergent, or cleaner.From the enzymatic properties of an enzyme, a conclusion cannot be madewithout problems on its contribution to the washing performance of acomposition. Here, further factors, for example, stability, substratebinding, binding to the articles to be cleaned or interactions withother constituents of the detergent or cleaner, in particular synergies,play a role in the removal of the soiling.

The amino acid sequences indicated in SEQ ID NO. 4 and 7 have, asdescribed in the examples of the present application, been derived fromnucleic acids which have been isolated from soil samples. Theirsequences are indicated under SEQ ID NO. 3 or 6. The derived proteinsare designated according to the invention as protease HP70 (for SEQ IDNO. 3 and 4) or HP53 (for SEQ ID NO. 6 and 7). As can be comprehendedwith the aid of alignment, for example by means of FIG. 4, they have ahomology to one another at the amino acid level of 93.9%.

An extracellular serine protease (E.C. 3.4.21.-) from Xanthomonascampestris pv. campestris (ATCC 33913), having GenBank (National Centerfor Biotechnology Information NCBI, National Institutes of Health,Bethesda, Md., USA) accession number NP_(—)636242 is the most similar tothe enzymes described herein. Another similar enzyme is an extracellularserine protease (E.C. 3.4.21.-) from Xanthomonas campestris pv.campestris (ATCC 33913), which in GenBank (National Center forBiotechnology Information NCBI, National Institutes of Health, Bethesda,Md., USA) carries the accession number NP_(—)636242. The homology ofthis enzyme, determined like all subsequent homology values by means ofthe computer program Vector NTI® Suite 7.0, obtainable from InforMax,Inc., Bethesda, USA, using the specified default parameters is, at theamino acid level, 75.0% identity to HP70 and 75.4% identity to HP53.

Other similar enzymes are compiled in Examples 4 and 5 in tabular form;they are each extracellular proteases from Xanthomonas campestris and X.axonopodis. A homology of 26.2% identity and at the nucleotide level of33.6% results in the established B. lentus alkaline protease (WO92/21760 A1) over the entire length of the alkaline protease HP70 at theamino acid level. HP53 has homology values of 25.9% and 33.5% identityto the B. lentus alkaline protease.

These data compiled according to the Examples can be updated in thefollowing way. The protease StmPr2 from St. maltophilia, (GenBank:AY253983) has a sequence homology of 84.7% to the protease HP70according to the invention (SEQ ID NO. 4) and of 82.5% identity to HP53(SEQ ID NO. 7). The protease disclosed under SEQ ID NO. 66 in WO2004/033668 A2 is 83.1% identical to HP70 and 81.1% identical to HP53.Compared to the protease disclosed under SEQ ID NO. 70 in WO 2004/033668A2, homology values of 85.0% identity to HP70 and of 82.3% identity toHP53 result.

Alkaline proteases within the scope of the invention are encoded by SEQID NO. 4, or sequences which are at least 90% identical thereto, SEQ IDNO. 7 or sequences which are at least 87.5% identical thereto.

Among these, functional alkaline proteases are preferred.

Increasingly preferred are all alkaline proteases of this type whoseamino acid sequences are identical to the amino acid sequence indicatedin SEQ ID NO. 4 to at least 95% and increasingly preferably to at least96%, 97%, 98%, 99% and very particularly preferably to 100% identical orto the amino acid sequence indicated in SEQ ID NO. 7, includingsequences which are at least 90% and increasingly preferably to at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and very particularlypreferably to 100% identical thereto where in each case all integral orfractional intermediate values are correspondingly included.

The associated vectors described in the examples, coding for theproteins which are derived from the vector shown in FIG. 3, were giventhe designations 70-pUC(AWB403) for HP70 and 53-pUC(AWB403) for HP53.They were deposited under this name on Oct. 2, 2003 at the DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg 1b,38124 Brunswick and there carry the deposit numbers DSM 15977 and DSM15976 respectively. The respective viability was confirmed by the DSMZon Oct. 17, 2003. The proteases encoded by these vectors, investigatedin the examples of the present application and therefore most stronglypreferred, are designated as HP70 and HP53 respectively.

Those alkaline proteases according to the invention also encompassfragments of the sequences which correspond to the amino acid positions33 to 581 as in SEQ ID NO. 4 or 39 to 586 as in SEQ ID NO. 7.

The mature protein is included above as the mature forms exert theindustrially relevant functions. Currently, it still cannot be saidwithout doubt which amino acid in each case represents the N-terminus ofthe mature protein. At the present time, the beginning in positions 33or 39 only appears most probable. Should it turn out at a later point intime that other amino acids represent the respective N-terminus, theclaimed field of protection will be based thereon, where the positionmentioned in each case designates the first amino acid of the matureprotein.

A sequence comparison of these mature enzymes carried out as in example4 with the nearest similar ones from the prior art has yielded thefollowing result: the (presumably) mature protease HP70 (SEQ ID NO. 4,positions 33 to 581) is identical to the homologous region of SEQ ID NO.66 from WO 2004/033668 A2 to 84.2%, to that of SEQ ID NO. 70 from WO2004/033668 A2 to 86.2% and to that of the protease STmPr2 to 85.8%. The(presumably) mature protease HP53 (SEQ ID NO. 7, positions 39 to 586) isidentical to the homologous region of SEQ ID NO. 66 from WO 2004/033668A2 to 83.8%, to that of SEQ ID NO. 70 from WO 2004/033668 A2 to 85.0%and to that of the protease STmPr2 to 85.2%.

The same applies for the C-terminus. At present, positions 581 and 586as in SEQ ID NO. 4 and 7 appear to be the C-termini, because thenucleotide positions 1744 to 1746 as in SEQ ID NO. 3 and the positions1759 to 1761 as in SEQ ID NO.6 in each case represent a stop codon.Should it turn out at a later point in time, however, that as a resultof processing another amino acid represents the C-terminus of themature, active protein, the claimed scope of protection will be relatedthereto, where the numbers indicated in each case designate the lastamino acid of the mature, active protein. In principle, the same appliesfor the case where on maturation of the protein internal fragments arepossibly excised. The amino acid sequence of the mature, active proteinis in each case particularly preferred.

Any of the alkaline proteases described up to now is furthermorepreferred in which the homology values from at least 90% identity ineach case apply for the region which corresponds to the amino acidpositions 33 to 470 as in SEQ ID NO. 5 or 33 to 470 as in SEQ ID NO. 8.

As is described in the Examples, the C-terminal regions mentionedtherein and hereby excluded from the preferred scope of protection couldbe deleted both from HP70 and from HP53 without the deletion variantslosing their protease activity, in particular, the proteolytic activityneeded during the washing or cleaning process. The advantage in thisdrastic deletion consists in the saving of expenditure and costs in thebiotechnological preparation of the proteins concerned. Thus, in ashorter time more enzymes usable according to the invention,particularly for use in detergents and cleaners, are obtained, which isalso accompanied, for example, by a better utilization of the mediaconstituents necessary for the fermentation of the producingmicroorganisms.

A sequence comparison of these mature and C-terminally deleted enzymeswith the nearest similar ones from the prior art carried out as inExample 4 has yielded the following result: the (presumably) mature andC-terminally deleted protease HP70 (SEQ ID NO. 5, positions 33 to 470)is identical to the homologous region of SEQ ID NO. 66 from WO2004/033668 A2 to 85.2%, to that of SEQ ID NO. 70 from WO 2004/033668 A2to 88.1% and to that of the protease STmPr2 to 87.7%. The (presumably)mature and C-terminally deleted protease HP53 (SEQ ID NO. 8, positions33 to 470) is identical to the homologous region of SEQ ID NO. 66 fromWO 2004/033668 A2 to 85.4%, to that of SEQ ID NO. 70 from WO 20041033668A2 to 87.0% and to that of the protease STmPr2 likewise to 87.0%.

Any of the alkaline proteases described up to now having an amino acidsequence as in the consensus sequence of SEQ ID NO. 9, preferably in therange of the amino acid positions 39 to 587, particularly preferably inthe range of the amino acid positions 39 to 476, is furthermorepreferred.

SEQ ID NO. 9 represents the consensus sequence obtainable from the twoamino acid sequences SEQ ID NO. 4 and 7, as can be established, forexample, by means of the alignment of FIG. 4. It comprises thoseproteases whose amino acid sequences in each of their position can betraced back either to SEQ ID NO. 4 or SEQ ID NO. 7. These two sequencesthus provide sequence information for subtilisin proteases related to orsimilar to one another. They have the general sequence indicated in SEQID NO. 9, where in the following 35 positions two different amino acidsin each case can be present or a certain amino acid (indicated in thethree-letter code) or no (−) amino acid; to be precise the followingpossibilities (in the sequence protocol in each case defined as a“variant”): position 2: —or Ile, position 3: Ser or Thr, position 4: Hisor Asn, position 5: Asp or Ser, position 7: —or Ser, position 8: —orVal, position 9: — or Pro, position 10: —or Gly, position 11: —or Asp,position 12: Gln or Pro, position 13: Pro or Gin, position 25: Ala orGly, position 48: Ser or Ala, position 65: Asn or Thr, position 66: Leuor Asp, position 82: Ser or Gin, position 149: Ala or Ser, position 234:Ser or Ala, position 236: Ile or Tyr, position 259: Ser or Thr, position267: Phe or Tyr, position 321: Thr or Ser, position 386: Ile or Val,position 406: Thr or Ala, position 438: Thr or Ser, position 487: Thr or—, position 488: Val or Thr, position 501: Ala or Ser, position 507: Seror Ala, position 511: Val or Ala, position 522: Ser or Thr, position527: Ser or Thr, position 546: Asn or Thr, position 562: Ser or Ala and,finally, position 574: Gly or Ala.

Since the two enzymes HP70 and HP53 in the investigations documented bythe present examples have advantages according to the invention andmoreover agree to 93.9%, it is to be expected that each further enzymewhich belongs to this protease subfamily has comparably favorableproperties.

This applies correspondingly to what has been said above, in particularfor the parts of the in each case mature, that is active, enzyme andvery particularly for those deletion variants in which large parts ofthe C-terminus are removed without noticeable loss of the proteaseactivity.

Each of the alkaline proteases described up to now is furthermorepreferred which is encoded by a nucleotide sequence which is identicalto the nucleotide sequence indicated in SEQ ID NO. 3 at least to 85% andincreasingly preferably to at least 90%, 95%, 96%, 97%, 98%, 99% andvery particularly preferably to 100%, in particular for the regioncorresponding to nucleotide positions 97 to 1746 as in SEQ ID NO. 3,very particularly for the region corresponding to nucleotide positions97 to 1410 as in SEQ ID NO. 3, or which is encoded by a nucleotidesequence which is identical to the nucleotide sequence indicated in SEQID NO. 6 at least to 85% and increasingly preferably to at least 90%,95%, 96%, 97%, 98%, 99% and very particularly preferably to 100%, inparticular for the region which corresponds to the nucleotide positions115 to 1761 as in SEQ ID NO. 6, very particularly for the regioncorresponding to nucleotide positions 115 to 1428 as in SEQ ID NO. 6,where in each case all integral or fractional intermediate values arecorrespondingly included.

As is explained in examples 3 and 4, the nearest similar enzyme to HP70and HP53 at the nucleotide level, an extracellular serine protease (E.C.3.4.21.-) from Xanthomonas campestris pv. campestris (ATCC 33913;NP_(—)636242), as can be determined by means of the computer programVector NTI® Suite 7.0, obtainable from InforMax, Inc., Bethesda, USA,using the specified default parameters, has a homology of 74.4 or 75.0%identity at the nucleotide level. Accordingly, all alkaline proteasesand proteins which are encoded by significantly more similar nucleicacids are included in the scope of protection.

These data compiled according to the examples can be updated in thefollowing way: The protease StmPr2 from St. maltophilia, (GenBank:AY253983) has a sequence homology of 80.8 at the DNA level to theprotease HP70 according to the invention (SEQ ID NO. 3) in thehomologizable region and to the DNA sequence of HP53 (SEQ ID NO. 6) of81.2% identity. The protease DNA sequence disclosed under SEQ ID NO. 65in WO 2004/033668 A2 is identical to that of HP70 to 79.6% and to thatof HP53 to 79.9%. In comparison to the protease-encoding DNA sequencedisclosed under SEQ ID NO. 69 in WO 2004/033668 A2, homology values of81.3% identity to the HP70 DNA and of 81.1% identity to the HP53 DNAresult.

The statements up to now apply correspondingly particularly for thenucleic acid sequences which code for the mature proteins and veryparticularly for the C-terminally deleted, proteolytically activevariants thereof. These are the enzymes whose proteolytic activities andin particular whose contributions to the washing or cleaning performanceof corresponding formulations are covered in the examples of the presentapplication.

A sequence comparison of these DNA sections coding for the matureenzymes carried out as in example 4 with the nearest similar ones fromthe prior art has yielded the following result: the gene for the(presumably) mature protease HP70 (SEQ ID NO. 3, positions 97 to 1746)is identical to the homologous region of SEQ ID NO. 65 from WO2004/033668 A2 to 80.0%, to that of SEQ ID NO. 69 from WO 2004/033668 A2to 81.8% and to that of the protease STmPr2 to 81.3%. The gene for the(presumably) mature protease HP53 (SEQ ID NO. 6, positions 115 to 1761)is identical to the homologous region of SEQ ID NO. 65 from WO2004/033668 A2 to 81.0%, to that of SEQ ID NO. 69 from WO 2004/033668 A2to 82.2% and to that of the protease STmPr2 to 82.9%.

The alkaline proteases derived therefrom are accordingly preferred.

A further sequence comparison of the DNA sections encoding for thesemature and C-terminally deleted enzymes is provided in example 4 withthe closest similar ones from the prior art yielding the followingresult: the gene for the (presumably) mature and C-terminally deletedprotease HP70 (SEQ ID NO. 3, positions 97 to 1410) is identical to thehomologous region of SEQ ID NO. 65 from WO 2004/033668 A2 to 81.4%, tothat of SEQ ID NO. 69 from WO 2004/033668 A2 to 83.7% and to that of theprotease STmPr2 to 83.2%. The nucleic acid encoding for the (presumably)mature and C-terminally deleted protease HP53 (SEQ ID NO. 6, positions115 to 1428) is identical to the homologous region of SEQ ID NO. 65 fromWO 2004/033668 A2 to 82.1%, to that of SEQ ID NO. 69 from WO 2004/033668A2 to 83.6% and to that of the protease STmPr2 to 83.9%.

The alkaline proteases encoding from these DNA sections are accordinglyparticularly preferred.

Furthermore, each of the alkaline proteases according to the inventiondescribed up to now, which is isolable from a natural habitat or whichis derived from a nucleic acid isolable from a natural habitat, ispreferred.

It is to be assumed that the DNAs isolated using the method described inthe examples have been formed from natural organisms and also code invivo for functional proteins. Thus, the associated enzymes themselvesmust also be able to be found by means of analogous methods, inparticular if they are not pseudogenes but actually formed proteins. Onthe other hand, the isolation of the nucleic acids leads immediately toa gene which can be introduced into molecular biologicalcharacterizations and produced. Moreover, it cannot always be expectedthat the genes concerned are expressed under all conditions, so thatnontranslated genes are also instantly accessible by means of thenucleic acid isolation.

Furthermore, each of the alkaline proteases according to the inventiondescribed up to now, which itself or whose associated nucleic acidoriginates from an organism which is isolable from a natural habitat, ispreferred.

This embodiment is therefore particularly advantageous, because then theassociated organism itself can be taken into culture. Advantageously,the proteases according to the invention can then be isolated andprepared from its cell extracts or culture supernatants.

Among these, those alkaline proteases are preferred where amicroorganism is involved, preferably a fungus or a bacterium, amongthese preferably a gram-positive bacterium and particularly preferablyone of the genus Bacillus.

Particularly for these organisms, culture methods are known andestablished in the prior art. This applies in particular for Bacilli,which take up an outstanding role in industrial enzyme production. Thosealkaline proteases and proteins which originate from Xanthomonas speciesare a further embodiment. From one of these gram-negative speciesoriginate the known enzymes determined as the nearest similar ones (seeabove); there is also already experience in the biotechnologicalfermentation of xanthomonads.

Alkaline proteases derived from one of the alkaline proteases accordingto the invention described up to now by fragmentation or deletionmutagenesis or proteins having at least 100 and increasingly preferablyat least 150, 200, 250 and very particularly preferably at least 300amino acids already connected in the starting molecule are furthermorepreferred.

Thus it is possible, for example, to delete individual amino acids atthe termini or in the loops of the enzyme without the proteolyticactivity being lost as a result. Such mutations are described, forexample, in WO 99/49057 A1. WO 01/07575 A2 teaches that by means of suchdeletions the allergenicity of proteases can be lowered and thus overalltheir employability can be improved. The fragmentation works to theadvantage of the insertion or substitution mutagenesis and/or fusioncarried out later with other enzymes. With respect to the intended useof these enzymes, it is preferred if they also have a proteolyticactivity after fragmentation or deletion mutagenesis; it is particularlypreferred if they have an activity additionally increased hereby.

Alkaline proteases or proteins as have been previously described asaccording to the invention and are derived from one of the alkalineproteases or proteins described up to now by insertion mutagenesis, bysubstitution mutagenesis and/or by fusion with at least one otherprotein are furthermore preferred.

Numerous documents from the prior art disclose advantageous effects ofinsertions and substitutions in proteases; among them also saidpublications WO 99/49057 A1 and WO 01/07575 A2. In principle, these alsoinclude individual replacements of amino acids; however, a number ofconnected amino acids can also be replaced by others. These also includenovel combinations of relatively large enzyme sections, that is theabove-mentioned fragments, with other proteases or proteins of otherfunction. Thus it is possible, for example, following WO 99/57254 A1, toprovide a protein according to the invention or parts thereof viapeptide linkers or directly as a fusion protein with binding domainsfrom other proteins, for example the cellulose binding domain, and as aresult to make the hydrolysis of the substrate more effective. Likewise,proteins according to the invention can, for example, also be linkedwith amylases or cellulases to exert a dual function.

Among these, the alkaline proteases or proteins having one or more aminoacid replacements in the positions 3, 4, 36, 42, 47, 56, 61, 69, 87, 96,99, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188,193, 199, 205, 211, 224, 229, 236, 237, 242, 243, 255 and 268 in thenumbering of the alkaline protease from Bacillus lentus are preferred,where these positions are to be assigned by means of the alignment inFIG. 1.

Here, the following amino acid residues lie in the wild-type molecule ofthe B. lentus alkaline protease: S3, V4, S36, N42, A47, T56, G61, T69,E87, A96, R99, A101, I102, S104, N114, H118, A120, S130, S139, T141,S142, S154, S157, A188, V193, V199, G205, L211, A224, K229, S236, N237,N242, H243, N255 and T268.

Since, in addition to the alkaline protease from Bacillus licheniformis,the B. lentus alkaline protease in the prior art is an importantreference molecule for the description of novel proteases and of pointmutations, the novel protease described here and thus also its sequencewere unknown up to now, it appears advantageous to refer to thisnumbering in the assignment of the point mutations. On the other hand,the numbering in general depends on the mature protein, and as mentionedabove it is still not definite at the present point in time with whichamino acid the mature protein begins. In the numbering of SEQ ID NO. 4(HP70), these positions—as can be understood by means of FIG.1—correspond to the following position numbers: P140, N141, T182, N188,Y195, A204, G209, T245, K264, K273, (−), Y277, T278, D280, V296, E304,I306, S317, G326, V328, S329, S341, V345, A376, S381, S393, G399, Y406,V419, Q424, T432, P433, T438, L439, G453 and V466.

In the numbering of SEQ ID NO. 7, that is HP53, these are the followingpositions: P146, N147, T188, N194, Y201, A210, G215, T251, K270, K279,(−), Y283, T284, D286, V302, E310, I312, S323, G332, V334, S335, S347,V351, A382, S387, S399, G405, Y412, V425, Q430, S438, P439, T444, L445,G459 and V472.

Thus, from the application WO 92/21760 A1 single and multiple variantsof the subtilisin from Bacillus lentus DSM 5483 in the followingpositions follow: 3, 4, 36, 42, 47, 56, 69, 87, 96, 101, 102, 104, 114,118, 120, 130, 139, 141, 142, 157, 188, 193, 199, 205, 224, 229, 236,237, 242, 243, 255 and 268. The application WO 95/23221 A1 additionallydiscloses replacements in this molecule in positions 99, 154 and 211, inparticular R99G, R99A, R99S, S154D, S154E, L211D and L211 E. On accountof the application WO 95/07770 A1, such variants are particularly alsosuitable for use in cosmetics. In addition to other replacements, thereplacement L211G is also described in the application WO 02/088340A2,and the replacement G61A in WO 03/038082 A2.

Among these, accordingly those are preferred in which the further aminoacid replacements are present in one or more of the positions 3, 4, 61,188, 193, 199 and 211. In HP70, the positions P140, N141, G209, A376,S381, S393 and Y406 correspond to this and in HP53 the positions P146,N147, G215, A382, S387, S399 and Y412.

Among these, corresponding to what has been said above, those are inturn preferred which are one or more of the amino acid replacements 3T,4I, 61A, 188P, 193M, 199I and 211D or 211G, providing thecorrespondingly homologous positions are not already naturally taken byone of these preferred amino acids.

The replacements S3T and V4I lead, as is explained in particular in WO02/088340 A2, presumably by means of a stabilizing effect to themolecule, to an improvement of its contribution to the washingperformance of a detergent or cleaner. The replacements S3T, V4I, A188P,V193M, V199I and L211D characterize the protease designated as F49according to WO 95/23221 A1, which has been used in Examples 7 and 8 ofthe present application as an efficient comparison enzyme established inthe prior art. On the other hand, the proteases HP70 and HP53 are stillunmodified wild-type molecules, whose activity, in particular theircontribution to the washing performance, might be improved by these samereplacements.

An alkaline protease according to the invention described beforehand orsuch a protein which is additionally stabilized is further preferred.

An increase in stability during storage and/or during use, for examplein the washing process, leads to its activity lasting longer and thusbeing increased in action. As stabilization possibilities, allstrategies which are described and expedient in the prior art aresuitable, for example, according to U.S. Pat. No. 5,230,891, covalentcoupling to a polymer.

Stabilizations are preferred which are possible by means of pointmutagenesis of the molecule itself. These necessitate, following theprotein recovery, no further working steps. Some point mutationssuitable for this are known per se from the prior art. Thus, accordingto U.S. Pat. Nos. 6,087,315 and 6,110,884, proteases can be stabilizedby replacing certain tyrosine residues for others.

Further possibilities are, for example:

-   -   modification of the binding of metal ions, in particular of the        calcium binding sites, for example according to the teaching of        the applications WO 88/08028 A1 and WO 88/08033 A1; according to        the first of these specifications one or more of the amino acid        residues involved in the calcium binding must be replaced by        negatively charged amino acids; according to the teaching of the        application WO 88/08033, for stabilization by means of the        calcium binding, point mutations must be introduced        simultaneously into at least one of the sequences of the two        radicals arginine/glycine;    -   according to the U.S. Pat. No. 5,453,372, proteins can be        protected against the influence of denaturing agents such as        surfactants by certain mutations on the surface.

Another possibility for stabilization with regard to increasedtemperature and the action of surfactants would be, in application ofthe teaching of WO 92/21760 A1, WO 02/088340 A2 and WO 03/038082 A2,stabilization by means of the replacement of amino acids which lie nearthe N-terminus by those which presumably come into contact with theremainder of the molecule by means of noncovalent interactions and thusmake a contribution to the maintenance of the globular structure. Thisis in particular advisable for alkaline proteases which have originallybeen obtained as B. lentus. Appropriate mutants having the variants asin SEQ ID NO. 12 and 16 are described in the examples of the presentapplication.

Preferred embodiments are those in which the molecule is stabilized in anumber of ways. For example, according to WO 89/09819 A1 it can beassumed that a number of stabilizing mutations act additively.

An alkaline protease according to the invention described beforehand orsuch a protein which is additionally derivatized is further preferred.

Derivatives are understood as meaning those proteins which are derivedby means of an additional modification of the proteins produced. Suchmodifications can, for example, influence the stability, substratespecificity or the binding strength to the substrate or the enzymaticactivity. They can also serve to reduce the allergenicity and/orimmunogenicity of the protein and thus, for example, to increase itsskin compatibility.

Such derivatizations can be carried out, for example, biologically, forexample in connection with the protein biosynthesis by the producinghost organism. Here, couplings of low molecular weight compounds such asof lipids or oligosaccharides are particularly to be emphasized.

Derivatizations, however, can also be carried out chemically, forexample by the chemical conversion of a side chain or by covalentbonding of another, for example macromolecular, compound to the protein.A chemical modification is described, for example, in the application DE4013142 A1. For example, the coupling of amines to carboxyl groups of anenzyme for the modification of the isoelectric point follows from WO95/26398 A1. For example, macromolecules such as proteins can be bondedto proteins according to the invention, for example by means ofbifunctional chemical compounds. For example, using the teaching of WO99/57154 A1 it is possible to provide a protein according to theinvention with a specific binding domain also by means of a non-proteinlinker. Such derivatives are particularly suitable for use in detergentsor cleaners. Analogously to WO 00/01831 A2, protease inhibitors can alsobe bonded to the proteins according to the invention via linkers, inparticular amino acid linkers. Couplings with other macromolecularcompounds, such as, for example, polyethylene glycol, improve themolecule with respect to further properties such as stability or skincompatibility; this has already been explained.

Derivatives of proteins according to the invention can in the widestsense also be understood as meaning preparations of these enzymes.Depending on obtainment, workup or preparation, a protein can beassociated with various other substances, for example from the cultureof the producing microorganisms. A protein can also have been treated,for example for increasing its storage stability, specifically withcertain other substances. All preparations of a protein according to theinvention are therefore also according to the invention. This is alsoindependent of whether it actually displays this enzymatic activity ornot in a certain preparation. It can be desirable that during storage ithas no or only low activity, and only displays its proteolytic functionat the time of use. This can be controlled, for example, by means ofappropriate concomitant substances. In particular, the joint preparationof proteases with protease inhibitors is advantageous and is known fromthe prior art (WO 00/01826 A2).

An alkaline protease according to the invention described beforehand orsuch a protein which has at least one antigenic determinant in commonwith one of the alkaline proteases or proteins designated beforehand, inparticular over at least one of the epitope regions within which thepositions 3, 4, 36, 42, 47, 56, 61, 69, 87, 96, 99, 101, 102, 104, 114,118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 211, 224,229, 236, 237, 242, 243, 255 and 268 lie in the numbering of thealkaline protease from Bacillus lentus, to be assigned by means of thealignment in FIG. 1, is further preferred.

This applies in particular for the variants in these positions describedabove, since on the one hand they are preferred per se and on the otherhand they can be differentiated by means of antibodies which have beenspecifically formed against these regions from the proteases which agreein these positions with the wild-type molecule.

The achievement of a subtask and thus an independent subject of theinvention are nucleic acids having a nucleotide sequence which isidentical to the nucleotide sequence indicated in SEQ ID NO. 3 at leastto 85% or to the nucleotide sequence indicated in SEQ ID NO. 6 at leastto 85%.

On the one hand, the detection of the protease described in the examplesis based on the isolation of the associated DNA. On the other hand, thenucleic acids can be immediately cloned and thus incorporated into therecombinant DNA production of the directed enzymes.

Among these, those are increasingly preferred which are identical to oneof the indicated nucleotide sequences increasingly preferably to atleast 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and veryparticularly preferably to 100%, where in each case all integral orfractional intermediate values are included.

Corresponding to the remarks made above and as is described in theexamples, as the nearest similar nucleotide sequences to SEQ ID NO. 3and SEQ ID NO. 6 only those having 74.4% and 75.0% identity could befound in the prior art.

The further DNA sequences following from WO 2004/033668 A2 and theGenBank entry AY253983 have already been discussed above and aresufficiently different to the nucleotide sequences according to theinvention described here.

Those nucleic acids according to the invention in which the homologyvalues in each case apply for the region which corresponds to thenucleotide positions 97 to 1746 as in SEQ ID NO. 3 or to the nucleotidepositions 115 to 1761 as in SEQ ID NO. 6 are further-more preferred.

Corresponding to what has been said above, the region is hereby meantwhich codes for the in each case mature, that is active, protein. Thestop codon is also included because its existence makes sure that alarger possibly no longer functional, unintentional fusion protein isnot formed. Thus, in the cloning, care is to be taken that a stop codonlikewise lies in this position, if a protein fusion is not specificallyto be caused by means of the C-terminus. Should it later turn out thatthe mature protein is formed from another part of this sequence, thescope of protection correspondingly applies for this part.

Those nucleic acids according to the invention are furthermore preferredin which the homology values in each case apply for the region whichcorresponds to the nucleotide positions 97 to 1410 as in SEQ ID NO. 3 orto the nucleotide positions 115 to 1428 as in SEQ ID NO. 6.

As is described in the examples, it suffices in the case of the proteinsHP70 and HP53 to use the N-terminal part of the total protein; aconsiderable C-terminal deletion in both cases led to an enzymeproteolytically active in detergents and cleaners, which has provided acorresponding contribution to the total cleaning performance of thecomposition concerned. Nucleic acids which code for such variants aretherefore preferred embodiments, because they make possible a morecost-efficient biotechnological preparation of the proteins concerned.

Furthermore and corresponding to the previous remarks, those nucleicacids according to the invention are preferred which code for analkaline protease or a protein of the first subject of the invention.

The same proteins should be made available by the present application,so that nucleic acids which code for only inactive proteins are not asolution according to the invention. Those nucleic acids which code formature proteins are preferred and, increasingly particularly, thosewhich code for increasingly more active variants.

Those nucleic acids according to the invention of which one orpreferably more codons are replaced by synonymous codons are furthermorepreferred.

This aspect relates in particular to the heterologous expression of theproteases concerned. Thus each organism, in particular each productionstrain has a certain codon usage. Here, bottlenecks in the proteinbiosynthesis can occur if the codons lying on the transgenic nucleicacid in the host cell are opposite to a comparatively small number ofloaded tRNAs. Synonymous codons code, on the other hand, for the sameamino acids and can be better translated, depending on the host. Thisoptionally necessary transcription thus depends on the choice of theexpression system. In particular with samples from unknown, possibly notculturable organisms, a corresponding adjustment may be necessary.

Corresponding to the remarks made above, the cells of an organism in thescope of protection are furthermore included and are an individualsubject of the invention, which naturally contains a nucleic acidaccording to the invention.

By means of their culturing, the desired enzymes can be directlyaccessible.

Particularly preferred among these are those cells which naturallyexpress and preferably secrete a protease or a protein of the firstsubject of the invention.

By means of this, proteases according to the invention can beimmediately tested with respect to their intended area of applicationand possibly obtained in relatively large amounts by immediate culturingof this organism.

Among these, in turn, those cells are preferred which aremicroorganisms, preferably fungi or bacteria, among them preferablygram-positive bacteria and particularly preferably those of the genusBacillus or gram-negative bacteria of the genus Xanthomonas.

With microorganisms, there has been in the prior art extensiveexperience with respect to the molecular biological techniques and theproduction. This applies particularly for gram-positive bacteria, ofwhich those of the genus Bacillus belong to the most familiar productionstrains. No less preferred, however, are gram-negative bacteria of thegenus Xanthomonas, which up to now were utilized in particular for theproduction of the extracellular polysaccharide xanthan. On account ofthe homology comparisons already discussed and shown in the examples, itmoreover appears possible that strains of this genus naturally producethe particularly preferred proteases according to the invention HP70 andHP53. At least, their production in closely related strains should beparticularly advantageously realizable, for example as far as theircodon usage is concerned.

A further independent subject of the invention are methods for theidentification of an alkaline protease of the first subject of theinvention, which are based on the isolation of a nucleic acid from anaturally colonized habitat.

As is confirmed by the present invention, for the identification ofnovel proteases it is not absolutely necessary also to isolate theproteases and microorganisms concerned from nature. In particular bymeans of shotgun cloning or alternatively by means of PCR primers forknown sequence motifs it is possible to discover the nucleic acidsconcerned directly. Such a method is presented in examples 1 to 3 of thepresent application. Accordingly, it is possible, for example, toculture the microorganism flora of soil samples, to isolate DNAtherefrom and by means of cloning in an expression vector to test forprotease expression.

Among said methods, those are preferred in which one, preferably two,oligonucleotides corresponding to one another are employed, which canserve as PCR primers and are derived from one of the two sequences SEQID NO. 3 or 6.

A comparable approach based on a PCR using suitable primers follows, forexample, from the application WO 03/002711 A2 in the example ofα-amylases. Thus, it is possible, instead of culturing themicroorganisms and preparing the DNA therefrom, directly to amplify thenucleic acids contained in a soil sample. For this, PCR-based approachesare suitable. In this case, the nucleotide sequences indicated under SEQID NO. 3 and 6 can serve as a prototype for the design of correspondingPCR primers. It is advantageous here to design primers according tomethods known per se, which exclusively or in particular N-terminalcomprise only little more than the mature protein; moreover, theknowledge obtained on the dc variants (Example 6) can be utilized to theeffect that by means of the PCR only nucleic acids coding forcorrespondingly truncated proteins are amplified.

Furthermore, those methods are preferred in which the isolated nucleicacid is cloned, preferably expressed and particularly preferablyidentified as a protease by means of the protease activity of theexpression product.

Cloning usually represents, even if a PCR has not been carried outbeforehand, the essential molecular biological step by which theobtainment of the associated enzyme is initiated. Expression serves forthe biochemical characterization of the protein derived from the nucleicacid. In particular, if the test for protease activity, for example bymeans of the degradation of a protein substrate (compare examples) issuccessful, it is possible to be certain to have found a protease, whichcan be investigated in subsequent tests with respect to its industrialusability.

A further independent subject of the invention are vectors which containa nucleic acid region according to the invention designated beforehand.

In order to deal with the nucleic acids relevant to the invention, andthus in particular to prepare for the production of proteins accordingto the invention, they are suitably ligated in vectors. Such vectors andthe associated working methods are described in detail in the prior art.Vectors are commercially obtainable in great number and variation width,both for cloning and for expression. These include, for example, vectorswhich are derived from bacterial plasmids, from bacteriophages or fromviruses, or mainly synthetic vectors. Furthermore, they aredifferentiated by the kind of cell types in which they are able to beestablished, for example by vectors for gram-negative, for gram-positivebacteria, for yeasts or for higher eukaryotes. They form suitablestarting points, for example, for molecular biological and biochemicalinvestigations and for the expression of the gene concerned orassociated protein.

In one embodiment, vectors according to the invention are cloningvectors.

Cloning vectors are suitable, in addition to the storage, the biologicalamplification or the selection of the gene of interest, for itsmolecular biological characterization. Simultaneously, they aretransportable and storable forms of the claimed nucleic acids and arealso starting points for molecular biological techniques, which are nottied to cells, such as, for example, the PCR or in vitro mutagenesismethods.

Preferably, vectors according to the invention are expression vectors.

Such expression vectors are the basis for producing the correspondingnucleic acids in biological production systems and therewith producingthe associated proteins. Preferred embodiments of this subject of theinvention are expression vectors which carry the genetic elementsnecessary for expression, for example the natural promoter, originallylocated upstream of this gene, or a promoter from another organism.These elements can be arranged, for example, in the form of an“expression cassette”. Alternatively, individual or all regulationelements can also be made available by the respective host cell.Particularly preferably, the expression vectors are matched with respectto further properties, such as, for example, the optimal copy number,with the chosen expression system, in particular the host cell (seebelow).

An independent subject of the invention are cells which afterrecombinant DNA modification contain one of the nucleic acid regionsaccording to the invention designated beforehand.

These cells contain the genetic information for the synthesis of aprotein according to the invention. Among them, in contrast to thenatural producers likewise claimed described above, those cells aremeant which according to methods known per se have been provided withthe nucleic acids according to the invention, or which are derived fromsuch cells. For this, suitably those host cells are selected which canbe cultured comparatively simply and/or produce high product yields.

They make possible, for example, the amplification of the correspondinggenes, but also their mutagenesis or transcription and translation andfinally the biotechnological production of the proteins concerned. Thisgenetic information can either be present extrachromosomally as aseparate genetic element that is in bacteria in a plasmidal location, orintegrated into a chromosome. The choice of a suitable system depends onquestions such as, for example, the type and duration of the storage ofthe gene, or of the organism or the type of mutagenesis or selection.For instance, mutagenesis and selection methods for the development ofdetergent enzymes based on bacteriophages—and their specific hostcells—are described in the prior art (WO 97/09446 A1).

In the countries where appropriate national laws demand that humanembryonic stem cells are excluded from such an application subject, thepresent invention is only claimed for a correspondingly restrictedsubject.

Preferably, said nucleic acid region lies on one of the vectorsaccording to the invention designated above, in particular on a cloningor expression vector.

They hereby become relevant to the realization of the present invention.

Furthermore, those cells are preferred which express, preferablysecrete, an alkaline protease or a protein of the first subject of theinvention.

Protein-forming host cells make possible their biotechnologicalproduction. Suitable host cells for protein expression are in principleall organisms, that is prokaryotes, eukaryotes or Cyanophyta. Those hostcells are preferred which genetically can be readily handled, whichconcerns, for example, the transformation with the expression vector,its stable establishment and the regulation of the expression, forexample monocellular fungi or bacteria. Moreover, preferred host cellsare distinguished by good microbiological and biotechnologicalhandleability. This relates, for example, to easy culturability, highgrowth rates, low requirements for fermentation media and goodproduction and secretion rates for foreign proteins. Preferably,laboratory strains are chosen which are oriented to expression. Suchstrains are obtainable commercially or by means of generally accessiblestrain collections. Each protein according to the invention can in thisway theoretically be obtained from a large number of host organisms.From the abundance of various systems available according to the priorart, the optimal expression systems for the individual case must bedetermined experimentally.

Host cells are particularly advantageous which are themselvesprotease-negative and thus do not degrade proteins formed.

Preferred embodiments are those host cells which on account ofappropriate genetic elements are regulatable in their activity, forexample by controlled addition of chemical compounds, by modification ofthe culturing conditions or as a function of the respective celldensity. This controllable expression makes possible a very economicalproduction of the proteins of interest; it is realizable, for example,by means of an appropriate element on the vector concerned. Suitably,gene, expression vector and host cell are matched to one another, as faras, for example, the genetic elements necessary for expression (ribosomebinding site, promoters, terminators) or the codon usage are concerned.

Among these, host cells are preferred which are characterized in thatthey are bacteria.

Bacteria are distinguished by short generation times and low demands onthe culturing conditions. Inexpensive methods can thereby beestablished. Moreover, we have an extensive wealth of experience withbacteria in fermentation technology. Gram-negative or gram-positivebacteria can be suitable for a special production for the most differentreasons, to be determined experimentally in the individual case, such asnutrient sources, product formation rate, time need etc.

In a preferred embodiment, these are gram-negative bacteria, inparticular of the genera Escherichia coli, Klebsiella, Pseudomonas orXanthomonas, in particular strains of E. coli K12, E. coli B orKlebsiella planticola, and very particularly derivatives of the strainsEscherichia coli BL21 (DE3), E. coli RV308, E. coli DH5.alpha., E. coliJM109, E. coli XL-1 or Klebsiella planticola (Rf).

In gram-negative bacteria, such as, for example, E. coli, a large numberof proteins are secreted into the periplasmatic space. This can beadvantageous for special applications. In the application WO 01/81597A1, a method is disclosed according to which it is achieved that evengram-negative bacteria expel the expressed proteins. Such a system isalso suitable for the production of proteins according to the invention.The gram-negative bacteria mentioned as preferred are as a ruleaccessible easily, that is commercially or by means of public straincollections, and optimizable to specific production conditions ininteraction with other components likewise available in large numbersuch as, for example, vectors.

As mentioned above, Xanthomonas, but also Pseudomonas are, on account oftheir suspected relationship to the strains producing HP70 and/or HP53in vivo, promising host cells; not least also because of a presumablysimilar codon usage.

In an alternative, no less preferred embodiment, these are agram-positive bacterium, in particular one of the genera Bacillus,Staphylococcus or Corynebacterium, very particularly of the speciesBacillus lentus, B. licheniformis, B. amyloliquefaciens, B. subtilis, B.globigii or B. alcalophilus, Staphylococcus carnosus or Corynebacteriumglutamicum.

Gram-positive bacteria have, compared to gram-negative bacteria, thebasic difference of releasing secreted proteins immediately into thenutrient medium surrounding the cells, from which, if this is desired,the expressed proteins according to the invention can be purifieddirectly from the nutrient medium. Moreover, they are related oridentical to most of the organisms of origin for industrially importantsubtilisins and usually themselves form comparable subtilisins, so thatthey have a similar codon usage and their protein synthesis apparatus isnaturally accordingly oriented. A further advantage can consist in thefact that by means of this method a mixture of proteins according to theinvention can be obtained with the subtilisins formed endogenously fromthe host strains. Such a coexpression likewise follows from theapplication WO 91/02792. Should they not be desired, the protease genesnaturally present in the host cell must be inactivated permanently ortemporarily.

Host cells are further preferred which are eukaryotic cells, preferablyof the genus Saccharomyces.

Examples of these are fungi such as Actinomycetes or simply yeasts suchas Saccharomyces or Kluyveromyces. Thermophilic fungal expressionsystems are presented, for example, in WO 96/02653 A1. These areparticularly suitable for the expression of temperature-resistantvariants. The modifications which eukaryotic systems carry out,particularly in connection with protein synthesis, include, for example,the binding of low molecular weight compounds such as membrane anchorsor oligosaccharides. Oligosaccharide modifications of this type can bedesirable, for example, for lowering the allergenicity. Coexpressionwith the enzymes naturally formed from cells of this type, such as, forexample, cellulases, can also be advantageous.

An independent subject of the invention are methods for the productionof an alkaline protease or of a protein according to the first subjectof the invention.

This includes all methods for the production of a protein according tothe invention described above, for example chemical synthesis methods.

On the other hand, however, all molecular biological, microbiological,or biotechnological preparation methods established in the prior art,already discussed in individual aspects above, are preferred.

Preferably, these are methods which are carried out using a nucleic acidaccording to the invention designated above, preferably carried outusing a vector designated beforehand and particularly preferably using acell designated beforehand.

By means of said nucleic acids, in particular the nucleic acidsindicated in the sequence protocol under SEQ ID NO. 3 or 6, thecorrespondingly preferred genetic information is made available inmicrobiologically utilizable form, that is, for recombinant DNAproduction methods. Provision on a vector particularly successfullyutilizable by the host cell or by such cells themselves is increasinglypreferred. The production methods concerned are known per se to theperson skilled in the art.

On the basis of the associated nucleic acid sequences, embodiments ofthe present invention can also be cell-free expression systems in whichprotein biosynthesis in vitro is understood. All elements alreadymentioned above can also be combined to give novel methods to produceproteins according to the invention. In this case, for each proteinaccording to the invention a large number of combination possibilitiesof process steps are conceivable, so that optimal processes for eachactual individual case have to be determined experimentally.

Corresponding to what has been said above under the cell-associatedmethods, those are preferred in which the nucleotide sequence has beenadapted in one or preferably more codons to the codon usage of the hoststrain.

An independent subject of the invention are compositions which comprisean alkaline protease according to the invention described above.

Thereby, all types of compositions, in particular mixtures, recipes,solutions etc., whose employability is improved by addition of a proteinaccording to the invention described above, are included in the scope ofprotection of the present invention. Here, depending on the area of use,these can be, for example, solid mixtures, for example powderscontaining freeze-dried or encapsulated proteins, or gelatinous orliquid compositions. Preferred recipes contain, for example, buffersubstances, stabilizers, reaction partners and/or cofactors of theproteases and/or other ingredients synergistic with the proteases. Inparticular, among these are to be understood compositions for the useareas mentioned further below. Further use areas follow from the priorart and are presented, for example, in the handbook Industrial enzymesand their applications by H. Uhlig, (New York, Wiley-Verlag, 1998).

As a preferred embodiment, compositions are to be included in thissubject of the invention which are detergents or cleaners.

As is shown in the working examples of the present application, it wassurprisingly possible for detergents and cleaners containing a preferredprotease according to the invention to observe a performance increasecompared to the protease-free composition.

This subject of the invention includes all conceivable types ofcleaners, both concentrates and compositions to be used undiluted, foruse on the commercial scale, in a washing machine or a hand wash, orhand cleaning. These include, for example, detergents for textiles,carpets, or natural fibers, for which the designation detergent is usedaccording to the present invention. These also include, for example,washing-up liquids for dishwashers or manual washing-up liquids orcleaners for hard surfaces such as metal, glass, porcelain, ceramic,tiles, stone, lacquered surfaces, plastics, wood or leather; accordingto the present invention the designation cleaner is used for these.

Embodiments of the present invention include all administration formsestablished according to the prior art and/or all expedientadministration forms of the detergents or cleaners according to theinvention. These include, for example, solid, pulverulent, liquid,gelatinous or pasty compositions, optionally also consisting of a numberof phases, compressed or uncompressed; and further include, for example:extrudates, granules, tablets or pouches, packed both in large drums andin portions.

In addition to an alkaline protease of the subtilisin type according tothe invention, a detergent or cleaner according to the inventionoptionally contains, according to its area of use, further ingredientssuch as further enzymes, enzyme stabilizers, surfactants, for examplenonionic, anionic and/or amphoteric surfactants, and/or bleaches, and/orbuilders, and optionally further customary ingredients, which arementioned in more detail below.

In a preferred embodiment, the detergents or cleaners according to theinvention contain the alkaline proteases of the subtilisin typeaccording to the invention described above in an amount from 2 μg to 20mg, preferably from 5 μg to 17.5 mg, particularly preferably from 20 μgto 15 mg, very particularly preferably from 50 μg to 10 mg per gram ofthe composition. All integral and nonintegral values in each case lyingbetween these numbers are included.

The protease activity in compositions of this type can be determinedaccording to the method described in Tenside [Surfactants], Vol. 7(1970), pp. 125-132. It is accordingly indicated in PU (protease units).

In the comparison of the performances of two detergent enzymes, as, forexample, in the examples of the present application, a distinction mustbe made between protein-identical and activity-identical use. Inparticular in the case of recombinantly obtained preparations largelyfree of additional activity, protein-identical use is appropriate. Thusa statement is possible about whether the same amounts of protein—as ameasure of the yield of the fermentative production—lead to comparableresults. If the respective ratios of active substance to total protein(the values of the specific activity) diverge, an activity-identicalcomparison is to be recommended, because by this means the respectiveenzymatic properties are compared. Generally, it applies that a lowspecific activity can be compensated by addition of a relatively largeamount of protein. This is, in the end, an economic consideration.

A nonexhaustive compilation of important ingredients customary fordetergents and cleaners now follows. As an alternative orsupplementarily, further ingredients suitable for the respective purposecan be added.

As nonionic surfactants, preferably alkoxylated, advantageouslyethoxylated, in particular primary alcohols having preferably 8 to 18 Catoms and on average 1 to 12 mol of ethylene oxide (EO) per mole ofalcohol are employed, in which the alcohol radical can be linear orpreferably methyl-branched in the 2-position, or can contain linear andmethyl-branched residues in the mixture, i.e. as are customarily presentin oxoalcohol residues. In particular, however, alcohol ethoxylateshaving linear residues of alcohols of native origin having 12 to 18 Catoms, for example from coconut, palm, tallow fatty or oleyl alcohol,and on average 2 to 8 EO per mole of alcohol are preferred. Thepreferred ethoxylated alcohols include, for example, C₁₂₋₁₄ alcoholshaving 3 EO or 4 EO, C₉₋₁₁ alcohol having 7 EO, C₁₃₋₁₅ alcohols having 3EO, 5 EO, 7 EO or 8 EO, C₁₂₋₁₈ alcohols having 3 EO, 5 EO or 7 EO andmixtures of these, such as mixtures of C₁₂₋₁₄ alcohol having 3 EO andC₁₂₋₁₈ alcohol having 5 EO. The degrees of ethoxylation indicated arestatistical mean values, which for a specific product can be an integralor a fractional number. Preferred alcohol ethoxylates have aconcentrated homolog distribution (narrow range ethoxylates, NRE). Inaddition to these nonionic surfactants, also fatty alcohols having morethan 12 EO can be employed. Examples of these are tallow fatty alcoholhaving 14 EO, 25 EO, 30 EO or 40 EO.

A further class of preferably employed nonionic surfactants, which areemployed either as a sole nonionic surfactant or in combination withother nonionic surfactants, are alkoxylated, preferably ethoxylated orethoxylated and propoxylated fatty acid alkyl esters, preferably having1 to 4 carbon atoms in the alkyl chain, in particular fatty acid methylesters.

A further class of nonionic surfactants which can be advantageouslyemployed are the alkylpolyglycosides (APG). Employablealkylpolyglycosides satisfy the general formula RO(G)_(z), in which R isa linear or branched, in particular methyl-branched in the 2-position,saturated or unsaturated, aliphatic radical having 8 to 22, preferably12 to 18 C atoms and G is the symbol which stands for a glycose unithaving 5 or 6 C atoms, preferably for glucose. The degree ofglycosylation z here lies between 1.0 and 4.0, preferably between 1.0and 2.0 and in particular between 1.1 and 1.4. Linearalkylpolyglucosides, that is alkylpolyglycosides in which thepolyglycosyl radical is a glucose radical and the alkyl radical is ann-alkyl radical, are preferably employed.

Nonionic surfactants of the amine oxides type, for example N-coconutalkyl-N,N-dimethylamine oxide and N-tallow alkyl-N,N-dihydroxyethylamineoxide, and of the fatty acid alkanolamides type can also be suitable.The content of these nonionic surfactants preferably does not lie abovethat of the ethoxylated fatty alcohols, in particular is not more thanhalf thereof.

Further suitable surfactants are polyhydroxyfatty acid amides of theformula (II)—

in which RCO is an aliphatic acyl radical having 6 to 22 carbon atoms,R¹ is hydrogen, an alkyl or hydroxyalkyl radical having 1 to 4 carbonatoms and [Z] is a linear or branched polyhydroxyalkyl radical having 3to 10 carbon atoms and 3 to 10 hydroxyl groups. The polyhydroxyfattyacid amides are known substances, which can customarily be obtained byreductive amination of a reducing sugar with ammonia, an alkylamine oran alkanolamine and subsequent acylation with a fatty acid, a fatty acidalkyl ester or a fatty acid chloride.

The group consisting of the polyhydroxyfatty acid amides also includescompounds of the formula (III)—

in which R is a linear or branched alkyl or alkenyl radical having 7 to12 carbon atoms, R¹ is a linear, branched or cyclic alkyl radical or anaryl radical having 2 to 8 carbon atoms and R² is a linear, branched orcyclic alkyl radical or an aryl radical or an oxyalkyl radical having 1to 8 carbon atoms, where C₁₋₄ alkyl or phenyl radicals are preferred and[Z] is a linear polyhydroxyalkyl radical, whose alkyl chain issubstituted by at least two hydroxyl groups, or alkoxylated, preferablyethoxylated or propoxylated derivatives of this radical.

[Z] is preferably obtained by reductive amination of a reducing sugar,for example glucose, fructose, maltose, lactose, galactose, mannose orxylose. The N-alkoxy- or N-aryloxy-substituted compounds can beconverted, for example, to the desired polyhydroxyfatty acid amides byreaction with fatty acid methyl esters in the presence of an alkoxide asa catalyst.

Anionic surfactants employed are, for example, those of the typeconsisting of the sulfonates and sulfates. Possible surfactants of thesulfonate type are in this case preferably C₉₋₁₃ alkylbenzenesulfonates,olefinsulfonates, that is, mixtures of alkene- andhydroxyalkanesulfonates and disulfonates such as are obtained, forexample, from C₁₂₋₁₈ monoolefins having a final or internal double bondby sulfonation with gaseous sulfur trioxide and subsequent alkaline oracidic hydrolysis of the sulfonation products. Also suitable arealkanesulfonates which are obtained from C₁₂₋₁₈ alkanes, for example, bysulfochlorination or sulfoxidation with subsequent hydrolysis orneutralization. Likewise, the esters of α-sulfofatty acids (estersulfonates), for example the α-sulfonated methyl esters of thehydrogenated coconut, palm kernel or tallow fatty acids, are alsosuitable.

Further suitable anionic surfactants are sulfated fatty acid glycerolesters. Fatty acid glycerol esters are to be understood as meaning themono-, di- and triesters and their mixtures, as are obtained in thepreparation by esterification of a monoglycerol having 1 to 3 mol offatty acid, or in the transesterification of triglycerides having 0.3 to2 mol of glycerol. Preferred sulfated fatty acid glycerol esters hereare the sulfation products of saturated fatty acids having 6 to 22carbon atoms, for example capric acid, caprylic acid, caproic acid,myristic acid, lauric acid, palmitic acid, stearic acid or behenic acid.

Preferred alk(en)ylsulfates are the alkali metal and in particular thesodium salts of the sulfuric acid hemiesters of the C₁₂-C₁₈ fattyalcohols, for example of coconut fatty alcohol, tallow fatty alcohol,lauryl, myristyl, cetyl or stearyl alcohol or the C₁₀-C₂₀ oxo alcoholsand those hemiesters of secondary alcohols of these chain lengths.Furthermore preferred are alk(en)ylsulfates having said chain length,which contain a synthetic straight-chain alkyl radical, prepared on apetrochemical basis, which have an analogous breakdown behavior to theadequate compounds based on fatty chemical raw materials. From laundrytechnology interest, the C₁₂-C₁₆ alkyl sulfates and C₁₂-C₁₅ alkylsulfates and C₁₄-C₁₅ alkyl sulfates are preferred. 2,3-Alkyl sulfatesare also suitable anionic surfactants.

The sulfuric acid monoesters of the straight-chain or branched C₇₋₂₁alcohols ethoxylated with 1 to 6 mol of ethylene oxide, such as2-methyl-branched C₉₋₁₁ alcohols having on average 3.5 mol of ethyleneoxide (EO) or C₁₂₋₁₈ fatty alcohols having 1 to 4 EO, are also suitable.They are employed only in relatively small amounts in cleaners onaccount of their high foam behavior, for example in amounts up to 5% byweight, customarily from 1 to 5% by weight.

Further suitable anionic surfactants are also the salts ofalkylsulfosuccinic acid, which are also designated as sulfosuccinates oras sulfosuccinic acid esters, and the monoesters and/or diesters ofsulfosuccinic acid with alcohols, preferably fatty alcohols and inparticular ethoxylated fatty alcohols. Preferred sulfosuccinates containC₈₋₁₈ fatty alcohol radicals or mixtures of these. In particular,preferred sulfosuccinates contain a fatty alcohol radical, which isderived from ethoxylated fatty alcohols, which considered per se arenonionic surfactants (for description see above). Here, in turn,sulfosuccinates whose fatty alcohol radicals are derived fromethoxylated fatty alcohols having a concentrated homolog distributionare particularly preferred. Likewise, it is also possible to employalk(en)ylsuccinic acid preferably having 8 to 18 carbon atoms in thealk(en)yl chain or its salts.

Possible further anionic surfactants are in particular soaps. Saturatedfatty acid soaps, such as the salts of lauric acid, myristic acid,palmitic acid, stearic acid, hydrogenated erucic acid and behenic acidand in particular soap mixtures derived from natural fatty acids, forexample coconut, palm kernel or tallow fatty acids are suitable.

The anionic surfactants including the soaps can be present in the formof their sodium, potassium or ammonium salts and as soluble salts oforganic bases, such as mono-, di- or triethanolamine. Preferably, theanionic surfactants are present in the form of their sodium or potassiumsalts, in particular in the form of the sodium salts.

The surfactants can be contained in the cleaners or detergents accordingto the invention as a whole in an amount of from preferably 5% by weightto 50% by weight, in particular of 8% by weight to 30% by weight, basedon the finished composition.

Detergents or cleaners according to the invention can contain bleaches.Among the compounds serving as bleaches, which yield H₂O₂ in water,sodium percarbonate, sodium perborate tetrahydrate and sodium perboratemonohydrate have particular importance. Further utilizable bleaches are,for example, peroxopyrophosphates, citrate perhydrates and peracid saltsor peracids yielding H₂O₂, such as persulfates and persulfuric acid.Also utilizable is the urea peroxohydrate percarbamide, which can bedescribed by the formula H₂N—CO—NH₂.H₂O₂. In particular when using thecompositions for cleaning hard surfaces, for example in mechanicaldishwashing, they can, if desired, also contain bleaches from the groupconsisting of the organic bleaches, although their use in principle isalso possible in compositions for textile washing. Typical organicbleaches are the diacyl peroxides, such as, for example, dibenzoylperoxide. Further typical organic bleaches are the peroxy acids, whereas examples particularly the alkylperoxy acids and the arylperoxy acidsare mentioned. Preferred representatives are peroxybenzoic acid and itsring-substituted derivatives, such as alkylperoxybenzoic acids, but alsoperoxy-.alpha.-naphthoic acid and magnesium monoperphthalate, thealiphatic or substituted aliphatic peroxy acids, such as peroxylauricacid, peroxystearic acid, .epsilon.-phthalimidoperoxycaproic acid(phthalimidoperoxyhexanoic acid, PAP), o-carboxybenzamidoperoxycaproicacid, N-nonenylamidoperadipic acid and N-nonenylamidopersuccinates, andaliphatic and araliphatic peroxy-dicarboxylic acids, such as1,12-diperoxycarboxylic acid, 1,9-diperoxyazelaic acid, diperoxysebacicacid, diperoxybrassylic acid, the diperoxyphthalic acids,2-decyldiperoxybutane-1,4-dioic acid,N,N-terephthaloyldi(6-aminopercaproic acid) can be employed.

The content of bleach in the detergents or cleaners can be 1 to 40% byweight and in particular 10 to 20% by weight, where advantageouslyperborate monohydrate or percarbonate is employed.

When washing at temperatures of 60° C. and below, and in particularduring the wash pretreatment, in order to achieve an improved bleachingaction the compositions can also contain bleach activators. Bleachactivators employed can be compounds which under perhydrolysisconditions afford aliphatic peroxocarboxylic acids preferably having 1to 10 C atoms, in particular 2 to 4 C atoms, and/or optionallysubstituted perbenzoic acid. Suitable substances are those which carryO- and/or N-acyl groups of said C atom number and/or optionallysubstituted benzoyl groups. Polyacylated alkylenediamines are preferred,in particular tetraacetylethylenediamine (TAED), acylated triazinederivatives, in particular1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT), acylatedglycolurils, in particular 1,3,4,6-tetraacetylglycoluril (TAGU),N-acylimides, in particular N-nonanoyl-succinimide (NOSI), acylatedphenolsulfonates, in particular n-nonanoyl- orisononanoyloxybenzenesulfonate (n- or iso-NOBS), acylatedhydroxycarboxylic acids, such as triethyl O-acetylcitrate (TEOC),carboxylic acid anhydrides, in particular phthalic anhydride, isatoicanhydride and/or succinic anhydride, carboxylic acid amides, such asN-methyldiacetamide, glycolide, acylated polyhydric alcohols, inparticular triacetin, ethylene glycol diacetate, isopropenyl acetate,2,5-diacetoxy-2,5-dihydrofuran and the enol esters known from Germanpatent applications DE 196 16693 and DE 196 16 767 and acetylatedsorbitol and mannitol or their mixtures described in European patentapplication EP 0 525 239 (SORMAN), acylated sugar derivatives, inparticular pentaacetylglucose (PAG), penta-acetylfructose,tetraacetylxylose and octaacetyllactose and also acetylated, optionallyN-alkylated glucamine or gluconolactone, triazole or triazolederivatives and/or particulate caprolactams and/or caprolactamderivatives, preferably N-acylated lactams, for exampleN-benzoylcaprolactam and N-acetylcaprolactam, which are known from theinternational patent applications WO 94/27970, WO 94/28102, WO 94/28103,WO 95/00626, WO 95/14759 and WO 95/17498. The hydrophilicallysubstituted acylacetals known from German patent application DE 196 16769 and the acyllactams described in German patent application DE 196 16770 and international patent application WO 95/14075 are likewisepreferably employed. The combinations of conventional bleach activatorsknown from German patent application DE 44 43 177 can also be employed.Likewise, nitrile derivatives such as cyanopyridines, nitrilequats, forexample N-alkylammonium acetonitriles, and/or cyanamide derivatives canbe employed. Preferred bleach activators are sodium4-(octanoyloxy)benzenesulfonate, n-nonanoyl- or isononanoyloxybenzenesulfonate (n- or iso-NOBS), undecenoyl oxybenzenesulfonate(UDOBS), sodium dodecanoyloxy-benzenesulfonate (DOBS),decanoyl-oxybenzoic acid (DOBA, OBC 10) and/or dodecanoyloxybenzenesulfonate (OBS 12), and N-methylmorpholinum acetonitrile(MMA). Bleach activators of this type can be contained in the customaryrange of amounts of 0.01 to 20% by weight, preferably in amounts of 0.1to 15% by weight, in particular 1% by weight to 10% by weight, based onthe total composition.

In addition to the conventional bleach activators or in their place,“bleach catalysts” can also be contained. These substances arebleach-strengthening transition metal salts or transition metalcomplexes such as, for example, Mn-, Fe-, Co-, Ru- or Mo-salenecomplexes or -carbonyl complexes. Mn, Fe, Co, Ru, Mo, Ti, V and Cucomplexes with N-containing tripod ligands and Co-, Fe-, Cu- andRu-ammine complexes are also suitable as bleach catalysts, where thosecompounds are preferably to be employed which are described in DE19709284 A1.

Detergents or cleaners according to the invention as a rule contain oneor more builders, in particular zeolites, silicates, carbonates, organiccobuilders and—where no ecological reasons speak against their use—alsothe phosphates. The latter are in particular builders preferably to beemployed in cleaners for mechanical dishwashing.

Crystalline, laminar sodium silicates of the general formulaNaMSi_(x)O_(2x+1).yH₂O may be mentioned here, where M is sodium orhydrogen, x is a number from 1.6 to 4, preferably 1.9 to 4.0 and y is anumber from 0 to 20 and preferred values for x are 2, 3 or 4.Crystalline layer silicates of this type are described, for example inEuropean patent application EP 164514. Preferred crystalline layersilicates of the formula indicated are those in which M is sodium and xassumes the values 2 or 3. In particular, both β- and δ-sodiumdisilicates Na₂Si₂O₅.yH₂O are preferred. Commercially, compounds of thistype are found, for example, under the name SKS® (Clariant). Thus,SKS-6® is mainly a δ-sodium disilicate having the formula Na₂Si₂O₅.yH₂O,in the case of SKS-7® mainly the β-sodium disilicate. By reaction withacids (for example citric acid or carbonic acid), kanemite Na₂Si₂O₅.yH₂Ois formed from the δ-sodium disilicate, commercially under the namesSKS-9® and SKS-10® (Clariant). It can also be advantageous to employchemical modifications of these layer silicates. For example, thealkalinity of the layer silicates can be suitably influenced. Incomparison to δ-sodium disilicate, layer silicates doped with phosphateor with carbonate have modified crystal morphologies, dissolve morerapidly and in comparison to δ-sodium disilicate show an increasedcalcium-binding power. Thus layer silicates of the general empiricalformula xNa₂O.ySiO₂.zP₂O₅, in which the ratio x to y corresponds to anumber 0.35 to 0.6, the ratio x to z corresponds to a number from 1.75to 1200 and the ratio y to z corresponds to a number from 4 to 2800, aredescribed in the patent application DE 196 01 063. The solubility of thelayer silicates can also be increased by employing particularly finelydivided layer silicates. Compounds of the crystalline layer silicateswith other ingredients can also be employed. Here, in particularcompounds with cellulose derivatives, which have advantages in thedisintegrating action and are in particular employed in detergenttablets, and compounds with polycarboxylates, for example citric acid,or polymeric polycarboxylates, for example copolymers of acrylic acid,may be mentioned.

Also employable are amorphous sodium silicates having a modulusNa₂O:SiO₂ of 1:2 to 1:3.3, preferably of 1:2 to 1:2.8 and in particularof 1:2 to 1:2.6, which are solution-retarded and have secondary washproperties. The solution retardation compared to conventional amorphoussodium silicates can have been produced here in various ways, forexample by surface treatment, compounding, compaction/compression or byoverdrying. In the context of this invention, the term “amorphous” isalso understood as meaning “X-ray-amorphous”. This means that in X-raydiffraction experiments the silicates do not produce any sharp X-rayreflections, as are typical for crystalline substances, but at most oneor more maxima of the scattered X-ray radiation, which have a breadth ofa number of degree units of the diffraction angle. However, it can veryprobably even lead to particularly good builder properties if thesilicate particles in electron diffraction experiments produceindistinct or even sharp diffraction maxima. This is to be interpretedin such a way that the products have microcrystalline regions of thesize 10 to a few hundred nm, where values to at most 50 nm and inparticular to at most 20 nm are preferred. In particular,compressed/compacted amorphous silicates, compounded amorphous silicatesand overdried X-ray amorphous silicates are preferred.

An optionally employable, fine-crystalline, synthetic and boundwater-containing zeolite is preferably zeolite A and/or P. As zeolite P,zeolite MAP® (commercial product of Crosfield) is particularlypreferred. However, zeolite X and mixtures of A, X and/or P are alsosuitable. Also commercially obtainable and in the context of the presentinvention preferably employable is, for example, a cocrystallizate ofzeolite X and zeolite A (about 80% by weight zeolite X), which ismarketed by CONDEA Augusta S.p.A. under the trade name VEGOBOND AX® andcan be described by the formulanNa₂O.(1-n)K₂O.Al₂O₃.(2-2.5)SiO₂.(3.5-5.5)H₂O

Suitable zeolites have a mean particle size of less than 10 μm (volumedistribution; measuring method: Coulter counter) and preferably contain18 to 22% by weight, in particular 20 to 22% by weight, of bound water.

Of course, use of the generally known phosphates as builder substancesis also possible, provided a use of this type should not be avoided forecological reasons. Among the large number of commercially obtainablephosphates, the alkali metal phosphates, with particular preference ofpentasodium or pentapotassium triphosphate (sodium orpotassium-tripolyphosphate), have the greatest importance in thedetergent and cleaner industry.

Alkali metal phosphates is here the summary designation for the alkalimetal (in particular sodium and potassium) salts of the variousphosphoric acids, in which a distinction can be made betweenmetaphosphoric acids (HPO₃)_(n) and orthophosphoric acid H₃PO₄ inaddition to higher molecular weight representatives. The phosphates herecombine a number of advantages in themselves—they act as alkalicarriers, prevent lime deposits on machine parts or lime incrustationsin fabrics and moreover contribute to the cleaning performance.

Sodium dihydrogenphosphate, NaH₂PO₄, exists as a dihydrate (density 1.91gcm⁻³, melting point 60° C.) and as a monohydrate (density 2.04 gcm⁻³).Both salts are white, very readily water-soluble powders, which onheating lose the water of crystallization and at 200° C. turn into theweakly acidic diphosphate (disodium hydrogendiphosphate, Na₂H₂P₂O₇), athigher temperature into sodium trimetaphosphate (Na₃P₃O₉) and Maddrell'ssalt (see below). NaH₂PO₄ has an acidic reaction; it is formed whenphosphoric acid is adjusted with sodium hydroxide solution to a pH of4.5 and the mash is sprayed. Potassium dihydrogenphosphate (primary ormonobasic potassium phosphate, potassium biphosphate, KDP), KH₂PO₄, is awhite salt of density 2.33 gcm⁻³, has a melting point of 253° C.[decomposition with formation of potassium polyphosphate (KPO₃)_(x)] andis readily soluble in water.

Disodium hydrogenphosphate (secondary sodium phosphate), Na₂HPO₄, is acolorless, very readily water-soluble crystalline salt. It exists inanhydrous form and with 2 mol (density 2.066 gcm⁻³, loss of water at 95°C.), 7 mol (density 1.68 gcm⁻³, melting point 48° C. with loss of 5H₂O)and 12 mol of water (density 1.52 gcm⁻³, melting point 35° C. with lossof 5H₂O), becomes anhydrous at 100° C. and on relatively strong heatingturns into the diphosphate Na₄P₂O₇. Disodium hydrogenphosphate isprepared by neutralization of phosphoric acid with sodium carbonatesolution using phenolphthalein as an indicator. Dipotassiumhydrogenphosphate (secondary or dibasic potassium phosphate), K₂HPO₄, isan amorphous, white salt which is readily soluble in water.

Trisodium phosphate, tertiary sodium phosphate, Na₃PO₄, are colorlesscrystals, which as the dodecahydrate have a density of 1.62 gcm⁻³ and amelting point of 73-76° C. (decomposition), as the decahydrate(corresponding to 19-20% P₂O₅) a melting point of 100° C. and inanhydrous form (corresponding to 39-40% P₂O₅) a density of 2.536 gcm⁻³.Trisodium phosphate is readily soluble in water with an alkalinereaction and is prepared by evaporating a solution of exactly 1 mol ofdisodium phosphate and 1 mol of NaOH. Tripotassium phosphate (tertiaryor tribasic potassium phosphate), K₃PO₄, is a white, deliquescent,granular powder of density 2.56 gcm⁻³, has a melting point of 1340° C.and is readily soluble in water with an alkaline reaction. It is formed,for example, on heating Thomas's slag with coal and potassium sulfate.In spite of the relatively high price, in the cleaner industry the morereadily soluble, therefore highly active, potassium phosphates arewidely preferred compared to corresponding sodium compounds.

Tetrasodium diphosphate (sodium pyrophosphate), Na₄P₂O₇, exists inanhydrous form (density 2.534 gcm⁻³, melting point 988° C., also stated880° C.) and as a decahydrate (density 1.815-1.836 gcm⁻³, melting point94° C. with loss of water). Both substances are colorless crystalssoluble in water with an alkaline reaction. Na₄P₂O₇ is formed on heatingdisodium phosphate to >200° C. or by reacting phosphoric acid withsodium carbonate in the stoichiometric ratio and dehydrating thesolution by spraying. The decahydrate complexes heavy metal salts andhardness formers and therefore decreases the hardness of the water.Potassium diphosphate (potassium pyrophosphate), K₄P₂O₇, exists in theform of the trihydrate and is a colorless, hygroscopic powder having thedensity 2.33 gcm⁻³, which is soluble in water, the pH of the 1% strengthsolution being 10.4 at 25° C.

By condensation of NaH₂PO₄ or of KH₂PO₄, relatively high molecularweight sodium and potassium phosphates are formed, in which cyclicrepresentatives, the sodium or potassium metaphosphates and chain-liketypes, the sodium or potassium polyphosphates, can be differentiated. Inparticular for the latter, a large number of designations are in use:fusible or calcined phosphates, Graham's salt, Kurrol's and Maddrell'ssalt. All higher sodium and potassium phosphates are together designatedas condensed phosphates.

The industrially important pentasodium triphosphate (Na₅P₃O₁₀; sodiumtripoly-phosphate) is a nonhygroscopic, white, water-soluble salt whichis anhydrous or crystallizes with 6H₂O, of the general formulaNaO—[P(O)(ONa)—O]_(n)—Na where n=3. In 100 g of water, approximately 17g of the salt free of water of crystallization dissolve at roomtemperature, about 20 g at 60° C., around 32 g at 100° C.; after heatingof the solution at 100° C. for two hours approximately 8% oforthophosphate and 15% of diphosphate are formed by hydrolysis. In thepreparation of pentasodium triphosphate, phosphoric acid is reacted withsodium carbonate solution or sodium hydroxide solution in astoichiometric ratio and the solution is dehydrated by spraying.Similarly to Graham's salt and sodium diphosphate, pentasodiumtriphosphate dissolves many insoluble metal compounds (lime, soaps,etc.). Pentapotassium triphosphate, K₅P₃O₁₀ (potassiumtripolyphosphate), comes onto the market, for example, in the form of a50% strength by weight solution (>23% P₂O₅, 25% K₂O). The potassiumpolyphosphates are widely used in the detergent and cleaner industry.Furthermore, sodium potassium tripolyphosphates also exist, which arelikewise employable in the context of the present invention. These areformed, for example, when sodium trimetaphosphate is hydrolyzed usingKOH:(NaPO₃)₃+2KOH→Na₃K₂P₃O₁₀+H₂O

These are employable according to the invention precisely as sodiumtripolyphosphate, potassium tripolyphosphate or mixtures of these two;mixtures of sodium tripolyphosphate and sodium potassiumtripolyphosphate or mixtures of potassium tripolyphosphate and sodiumpotassium tripolyphosphate or mixtures of sodium tripolyphosphate andpotassium tripolyphosphate and sodium potassium tripolyphosphate arealso employable according to the invention.

Organic cobuilders which can be employed in the detergents and cleanersaccording to the invention are, in particular, polycarboxylates orpolycarboxylic acids, polymeric polycarboxylates, polyaspartic acid,polyacetals, optionally oxidized dextrins, further organic cobuilders(see below) and phosphonates. These classes of substance are describedbelow.

Usable organic builder substances are, for example, the polycarboxylicacids employable in the form of their sodium salts, polycarboxylic acidsbeing understood as meaning those carboxylic acids which carry more thanone acid function. For example, these are citric acid, adipic acid,succinic acid, glutaric acid, malic acid, tartaric acid, maleic acid,fumaric acid, sugar acids, aminocarboxylic acids, nitrilotriacetic acid(NTA), provided a use of this type is not to be avoided for ecologicalreasons, and mixtures of these. Preferred salts are the salts of thepolycarboxylic acids such as citric acid, adipic acid, succinic acid,glutaric acid, tartaric acid, sugar acids and mixtures of these.

The acids per se can also be employed. In addition to their builderaction, they typically also have the property of an acidificationcomponent and thus also serve for the setting of a relatively low andrelatively mild pH of detergents or cleaners, provided the pH resultingdue to the mixing of the other components is not desired. In particular,here system- and environmentally tolerable acids such as citric acid,acetic acid, tartaric acid, malic acid, lactic acid, glycolic acid,succinic acid, glutaric acid, adipic acid, gluconic acid and any desiredmixtures of these may be mentioned. However, mineral acids, inparticular sulfuric acid or bases, in particular ammonium or alkalimetal hydroxides can also serve as pH regulators. Regulators of thistype are contained in the compositions according to the invention inamounts of preferably not over 20% by weight, in particular of 1.2% byweight to 17% by weight.

As builders, further polymeric polycarboxylates are suitable, these are,for example, the alkali metal salts of polyacrylic acid or ofpolymethacrylic acid, for example those having a relative molecular massof 500 to 70,000 g/mol.

Within the meaning of this specification, the molar masses indicated forpolymeric polycarboxylates are weight-average molar masses Mw of therespective acid form, which were basically determined by means of gelpermeation chromatography (GPC), a UV detector being employed.Measurement was carried out here against an external polyacrylic acidstandard, which on account of its structural relationship with thepolymers investigated yields realistic molecular weight values. Thesedata differ distinctly from the molecular weight data in whichpolystyrenesulfonic acids are employed as a standard. The molar massesmeasured against polystyrenesulfonic acids are as a rule distinctlyhigher than the molar masses indicated in this specification.

Suitable polymers are in particular polyacrylates which preferably havea molecular mass of 2000 to 20,000 g/mol. On account of their superiorsolubility, from this group, in turn, the short-chain polyacrylates,which have molar masses of 2000 to 10,000 g/mol, and particularlypreferably of 3000 to 5000 g/mol, can be preferred.

Furthermore suitable are copolymeric polycarboxylates, in particularthose of acrylic acid with methacrylic acid and acrylic acid ormethacrylic acid with maleic acid. Copolymers of acrylic acid withmaleic acid have proven particularly suitable, which contain 50 to 90%by weight of acrylic acid and 50 to 10% by weight of maleic acid. Theirrelative molecular mass, based on free acids, is in general 2000 to70,000 g/mol, preferably 20,000 to 50,000 g/mol and in particular 30,000to 40,000 g/mol. The (co)polymeric polycarboxylates can be employedeither as a powder or as an aqueous solution. The content of(co)polymeric polycarboxylates in the compositions can be from 0.5 to20% by weight, in particular 1 to 10% by weight.

For an improvement of the water solubility, the polymers can alsocontain allylsulfonic acids, such as, for example,allyloxybenzenesulfonic acid and methallylsulfonic acid, as monomers.

In particular, biodegradable polymers of more than two different monomerunits are also preferred, for example those which as monomers containsalts of acrylic acid and of maleic acid and vinyl alcohol or vinylalcohol derivatives or which as monomers contain salts of acrylic acidand 2-alkylallylsulfonic acid and sugar derivatives.

Further preferred copolymers are those which as monomers preferablycontain acrolein and acrylic acid/acrylic acid salts or acrolein andvinyl acetate.

Likewise, further preferred builder substances which may be mentionedare polymeric aminodicarboxylic acids, their salts or their precursorsubstances. Polyaspartic acids and their salts and derivatives areparticularly preferred.

Further suitable builder substances are polyacetals which can beobtained by reaction of dialdehydes with polyolcarboxylic acids whichhave 5 to 7 C atoms and at least 3 hydroxyl groups. Preferredpolyacetals are obtained from dialdehydes such as glyoxal,glutaraldehyde, terephthalaldehyde and their mixtures and frompolyolcarboxylic acids such as gluconic acid and/or glucoheptonoic acid.

Further suitable organic builder substances are dextrins, for exampleoligomers and polymers of carbohydrates, which can be obtained bypartial hydrolysis of starches. The hydrolysis can be carried outaccording to customary methods, for example acid- or enzyme-catalyzedmethods. Preferably, they are hydrolysis products having average molarmasses in the range from 400 to 500,000 g/mol. Here, a polysaccharidehaving a dextrose equivalent (DE) in the range from 0.5 to 40, inparticular from 2 to 30, is preferred, where DE is a customary measureof the reducing action of a polysaccharide in comparison to dextrose,which has a DE of 100. Both maltodextrins having a DE between 3 and 20and dry glucose syrups having a DE between 20 and 37 and also “yellowdextrins” and “white dextrins” having relatively high molar masses inthe range from 2000 to 30,000 g/mol are usable.

The oxidized derivatives of dextrins of this type are their reactionproducts with oxidants which are able to oxidize at least one alcoholfunction of the saccharide ring to the carboxylic acid function.Particularly preferred organic builders for compositions according tothe invention are oxidized starches, or their derivatives from theapplications EP 472042, WO 97/25399, and EP 755944.

Oxydisuccinates and other derivatives of disuccinates, preferablyethylenediamine disuccinate, are also further suitable cobuilders. Here,ethylenediamine-N,N′-disuccinate (EDDS) is preferably used in the formof its sodium or magnesium salts. Furthermore, in this connectionglycerol disuccinates and glycerol trisuccinates are also preferred.Suitable use amounts in zeolite-, carbonate- and/or silicate-containingformulations lie between 3 and 15% by weight.

Further usable organic cobuilders are, for example, acetylatedhydroxycarboxylic acids and their salts, which can optionally also bepresent in lactone form and which contain at least 4 carbon atoms and atleast one hydroxyl group and also at most two acid groups.

A further substance class with cobuilder properties are thephosphonates. These are in particular hydroxyalkane or aminoalkanephosphonates. Among the hydroxyalkane phosphonates, 1-hydroxyethane1,1-diphosphonate (HEDP) is of particular importance as a cobuilder. Itis preferably employed as the sodium salt, the disodium salt having aneutral reaction and the tetrasodium salt an alkaline reaction (pH 9).Suitable aminoalkane phosphonates are preferably ethylenediaminetetramethylenephosphonate (EDTMP), diethylenetriaminepentamethylenephosphonate (DTPMP) and their higher homologs. They arepreferably employed in the form of the neutral-reacting sodium salts,e.g. as the hexasodium salt of EDTMP or as the hepta- and octasodiumsalt of DTPMP. As a builder, from the class of the phosphonatespreferably HEDP is used here. The aminoalkane phosphonates moreover havemarked heavy-metal binding power. Accordingly, in particular if thecompositions also contain bleach, it can be preferred to use aminoalkanephosphonates, in particular DTPMP, or mixtures of said phosphonates.

Moreover, all compounds which are able to form complexes with alkalineearth metal ions can be employed as cobuilders.

Builder substances can optionally be present in the detergents orcleaners according to the invention in amounts up to 90% by weight. Theyare preferably present in amounts up to 75% by weight. Detergentsaccording to the invention have builder contents of, in particular, 5%by weight to 50% by weight. In compositions according to the inventionfor cleaning hard surfaces, in particular for the mechanical cleaning ofdishes, the content of builder substances is in particular 5% by weightto 88% by weight, preferably no water-insoluble builder materials beingemployed in compositions of this type. In a preferred embodiment ofcompositions according to the invention for, in particular, mechanicalcleaning of dishes, 20% by weight to 40% by weight of water-solubleorganic builders, in particular alkali metal citrate, 5% by weight to15% by weight of alkali metal carbonate and 20% by weight to 40% byweight of alkali metal disilicate are present.

Solvents which can be employed in the liquid to gelatinous compositionsof detergents and cleaners originate, for example, from the groupconsisting of mono- or polyhydric alcohols, alkanolamines or glycolethers, provided they are miscible with water in the concentration rangeindicated. Preferably, the solvents are selected from ethanol, n- ori-propanol, butanols, ethylene glycol methyl ether, ethylene glycolethyl ether, ethylene glycol propyl ether, ethylene glycol mono-n-butylether, diethylene glycol methyl ether, diethylene glycol ethyl ether,propylene glycol methyl, ethyl or propyl ether, dipropylene glycolmonomethyl, or ethyl ether, diisopropylene glycol monomethyl, or ethylether, methoxy-, ethoxy- or butoxytriglycol, 1-butoxyethoxy-2-propanol,3-methyl-3-methoxybutanol, propylene glycol t-butyl ether and mixturesof these solvents.

Solvents can be employed in the liquid to gelatinous detergents andcleaners according to the invention in amounts between 0.1 and 20% byweight, but preferably below 15% by weight and in particular under 10%by weight.

For adjustment of the viscosity, one or more thickeners, or thickeningsystems, can be added to the composition according to the invention.These high molecular weight substances, which are also called swellingagents, usually absorb the liquids and swell here, in order finally toturn to viscous true or colloidal solutions.

Suitable thickeners are inorganic or polymeric organic compounds. Theinorganic thickeners include, for example, polysilicic acids, clayminerals such as montmorillonites, zeolites, silicas and bentonites. Theorganic thickeners originate from the groups consisting of the naturalpolymers, of the modified natural polymers and of the fully syntheticpolymers. Such polymers originating from nature are, for example,agar-agar, carrageenan, tragacanth, gum arabic, alginates, pectins,polyoses, guar flour, carob bean flour, starch, dextrins, gelatin andcasein. Modified natural substances which are used as thickenersoriginate especially from the group consisting of the modified starchesand celluloses. By way of example, carboxymethylcellulose and othercellulose ethers, hydroxyethyl- and -propylcellulose and pome flourethers may be mentioned here. Fully synthetic thickeners are polymerssuch as polyacrylic and polymethacrylic compounds, vinyl polymers,polycarboxylic acids, polyethers, polyimines, polyamides andpolyurethanes.

The thickeners can be present in an amount up to 5% by weight,preferably from 0.05 to 2% by weight, and particularly preferably from0.1 to 1.5% by weight, based on the finished composition.

The detergent and cleaner according to the invention can optionallycontain as further customary ingredients sequestering agents,electrolytes and further auxiliaries, such as optical brighteners,graying inhibitors, silver corrosion inhibitors, color transferinhibitors, foam inhibitors, abrasives, colorants and/or scents, andmicrobial active compounds, UV absorbents and/or enzyme stabilizers.

Textile detergents according to the invention can contain as opticalbrighteners derivatives of diaminostilbenedisulfonic acid or its alkalimetal salts. For example, salts of4,4′-bis(2-anilino-4-morpholino-1,3,5-triazinyl-6-amino)stilbene-2,2′-dis-ulfonicacid or similarly synthesized compounds are suitable which, instead ofthe morpholino group carry a diethanolamino group, a methylamino group,an anilino group or a 2-methoxyethylamino group. Furthermore,brighteners of the type consisting of the substituted diphenylstyrylscan be present, for example the alkali metal salts of4,4′-bis(2-sulfostyryl)diphenyl,4,4′-bis(4-chloro-3-sulfostyryl)diphenyl, or4-(4-chlorostyryl)-4′-(2-sulfostyryl)diphenyl. Mixtures of theabovementioned optical brighteners can also be used.

Graying inhibitors have the object of keeping the dirt detached from thetextile fibers suspended in the liquor. For this, water-solublecolloids, usually of organic nature, are suitable, for example starch,size, gelatin, salts of ether carboxylic acids or ether sulfonic acidsof starch or of cellulose or salts of acidic sulfuric acid esters ofcellulose or of starch. Water-soluble polyamides containing acidicgroups are also suitable for this purpose. Furthermore, otherderivatives than the abovementioned starch derivatives can be used, forexample aldehyde starches. Preferably, cellulose ethers such ascarboxymethylcellulose (Na salt), methylcellulose, hydroxyalkylcelluloseand mixed ethers, such as methylhydroxyethylcellulose,methylhydroxypropylcellulose, methylcarboxy-methylcellulose and theirmixtures, for example in amounts from 0.1 to 5% by weight, based on thecompositions are employed.

In order to effect silver corrosion protection, silver corrosioninhibitors can be employed in cleaners according to the invention fordishes. Such inhibitors are known from the prior art, for examplebenzotriazoles, iron (III) chloride or CoSO₄. As is known, for example,from European patent specification EP 0 736 084 B1, particularlysuitable silver corrosion inhibitors for joint use with enzymes aremanganese, titanium, zirconium, hafnium, vanadium, cobalt or ceriumsalts and/or complexes, in which said metals are present in one of theoxidation states II, III, IV, V or VI. Examples of compounds of thistype are MnSO₄, V₂O₅, V₂O₄, VO₂, TiOSO₄, K₂TiF₆, K₂ZrF₆, Co(NO₃)₂,Co(NO₃)₃, and their mixtures.

“Soil release” active compounds or “soil repellents” are usuallypolymers which on use in a detergent impart dirt-repellent properties tothe wash fibers and/or assist the dirt-removing power of the otherdetergent constituents. A comparable effect can also be observed intheir use in cleaners for hard surfaces.

Soil-release active compounds which are particularly effective and havebeen known for a long time are copolyesters with dicarboxylic acid,alkylene glycol and polyalkylene glycol units. Examples of these arecopolymers or mixed polymers of polyethylene terephthalate andpolyoxyethylene glycol (DT 16 17 141, and DT 22 00 911 respectively). InGerman laid-open specification DT 22 53 063, acidic compositions arementioned, which inter alia contain a copolymer of a dibasic carboxylicacid and an alkylene or cycloalkylene polyglycol. Polymers of ethyleneterephthalate and polyethylene oxide terephthalate and their use indetergents are described in German specifications DE 28 57 292 and DE 3324 258 and European patent specification EP 0 253 567. European patentEP 066 944 relates to compositions which contain a copolyester ofethylene glycol, polyethylene glycol, aromatic dicarboxylic acid andsulfonated aromatic dicarboxylic acid in specific molar ratios. FromEuropean patent EP 0 185 427, methyl or ethyl group end-closedpolyesters with ethylene and/or propylene terephthalate and polyethyleneoxide terephthalate units and detergents which contain soil-releasepolymers of this type are known. European patent EP 0 241 984 relates toa polyester which, in addition to oxyethylene groups and terephthalicacid units also contains substituted ethylene units and glycerol units.From European patent EP 0 241 985, polyesters are known which inaddition to oxyethylene groups and terephthalic acid units contain1,2-propylene, 1,2-butylene and/or 3-methoxy-1,2-propylene groups andglycerol units and are end group-closed with C₁- to C₄-alkyl groups.From European patent application EP 0 272 033, polyesters at leastpartly end group-closed by C₁₋₄ alkyl or acyl radicals withpolypropylene terephthalate and polyoxyethylene terephthalate units areknown. European patent EP 0 274 907 describes sulfoethyl endgroup-closed terephthalate-containing soil-release polyesters. Accordingto European patent application EP 0 357 280, by sulfonation ofunsaturated end groups, soil-release polyesters having terephthalate,alkylene glycol and poly-C₂₋₄-glycol units are prepared. Internationalpatent application WO 95/32232 relates to acidic, aromatic dirtremoval-empowering polyesters. From international patent application WO97/31085, nonpolymeric soil-repellent active compounds for materialsmade of cotton having a number of functional units are known: A firstunit which, for example, can be cationic, is capable of adsorption onthe cotton surface by electrostatic interaction, and a second unit,which is of hydrophobic design, is responsible for the active compoundremaining on the water/cotton interface.

The color transfer inhibitors suitable for use in textile detergentsaccording to the invention in particular include polyvinylpyrrolidones,polyvinylimidazoles, polymeric N-oxides such as poly(vinylpyridineN-oxide) and copolymers of vinylpyrrolidone with vinylimidazole.

When used in mechanical cleaning methods, it can be advantageous to addfoam inhibitors to the compositions concerned. Suitable foam inhibitorsare, for example, soaps of natural or synthetic origin, which have ahigh content of C₁₈-C₂₄ fatty acids. Suitable nonsurfactant-like foaminhibitors are, for example, organopolysiloxanes and their mixtures withmicrofine, optionally silanized silicic acid and paraffins, waxes,microcrystalline waxes and their mixtures with silanized silicic acid orbistearylethylenediamide. Mixtures of various foam inhibitors are alsoused to advantage, for example those consisting of silicones, paraffinsor waxes. Preferably, the foam inhibitors, in particular silicone-and/or paraffin-containing foam inhibitors, are bound to a granular,water-soluble, or -dispersible, carrier substance. In particular,mixtures of paraffins and bistearylethylenediamides are preferred.

A cleaner according to the invention for hard surfaces can moreovercontain constituents having abrasive action, in particular from thegroup comprising quartz powder, sawdust, plastic powder, chalks andmicroglass beads, and their mixtures. Abrasives are present in thecleaners according to the invention preferably not over 20% by weight,in particular from 5% by weight to 15% by weight.

Colorants and scents are added to detergents and cleaners in order toimprove the esthetic impression of the products and to make available tothe consumer, in addition to the washing and cleaning performance, avisually and sensorily “typical and unmistakable” product. Perfume oilsand scents which can be used are individual odorants, for example thesynthetic products of the type consisting of the esters, ethers,aldehydes, ketones, alcohols and hydrocarbons. Odorants of the typeconsisting of the esters are, for example, benzyl acetate, phenoxyethylisobutyrate, p-tert-butylcyclohexyl acetate, linalyl acetate,dimethylbenzylcarbinyl acetate, phenylethyl acetate, linalyl benzoate,benzyl formate, ethylmethylphenyl glycinate, allylcyclohexyl propionate,styrallyl propionate and benzyl salicylate. The ethers include, forexample, benzyl ethyl ether, the aldehydes, for example, the linearalkanals having 8-18 C atoms, citral, citronellal,citronellyloxyacetaldehyde, cyclamenaldehyde, hydroxycitronellal, lilialand bourgeonal, the ketones, for example, the ionones,.alpha.-isomethylionone and methyl cedryl ketone, the alcohols anethol,citronellol, eugenol, geraniol, linalool, phenylethyl alcohol andterpineol, the hydrocarbons mainly include the terpenes such as limoneneand pinene. Preferably, however, mixtures of various odorants are used,which together produce a pleasant scent note. Such perfume oils can alsocontain natural odorants, such as are accessible from plant sources, forexample pine, citrus, jasmine, patchouli, rose or ylang-ylang oil.Muscatel, sage oil, camomile oil, oil of cloves, melissa oil, mint oil,oil of cinnamon leaves, linden blossom oil, oil of juniper berries,vetiver oil, olibanum oil, galbanum oil and labdanum oil and also orangeblossom oil, neroliol, orange peel oil and sandalwood oil. Customarily,the content of colorants in detergents and cleaners lies below 0.01% byweight, while scents can make up up to 2% by weight of the totalformulation.

The scents can be incorporated directly into the detergents or cleaners,but it can also be advantageous to apply the scents to carriers whichincrease the adhesion of the perfume to the material to be cleaned andby means of a slower release of scent provide for a long-lasting scent,in particular from treated textiles. Such carrier materials which haveproven to be suitable are, for example, cyclodextrins, where thecyclodextrin-perfume complexes can additionally also be coated withfurther auxiliaries. A further preferred carrier for scents is thedescribed zeolite X, which can also absorb scents instead of or as amixture with surfactants. Detergents and cleaners which contain thedescribed zeolite X and scents which are preferably absorbed at leastpartly on the zeolite are therefore preferred.

Preferred colorants whose choice causes no difficulty at all to theperson skilled in the art have a high storage stability andinsensitivity to the other ingredients of the compositions and to light,and no marked substantivity to textile fibers, in order not to colorthese.

For the control of microorganisms, detergents or cleaners can containantimicrobial active compounds. Here, a distinction is made, dependingon the antimicrobial spectrum and mechanism of action, betweenbacteriostatics and bactericides, fungistatics and fungicides etc.Important substances from these groups are, for example, benzalkoniumchlorides, alkylarylsulfonates, halophenols and phenol mercuriacetate.In the context of the teaching according to the invention, the termsantimicrobial action and antimicrobial active compound have the meaningstandard in practice, which is given, for example, by K. H. Wallhäuβerin Praxis der Sterilisation, Desinfektion—Konservierung:Keimidentifizierung—Betriebshygiene[Practice of Sterilization,Disinfection—Preservation: Microorganism Identification—Plant Hygiene](5th ed., Stuttgart, New York: Thieme, 1995), where all substancesdescribed there having antimicrobial action can be employed. Suitableantimicrobial active compounds are preferably selected from the groupsconsisting of the alcohols, amities, aldehydes, antimicrobial acids ortheir salts, carboxylic acid esters, acid amides, phenols, phenolderivatives, diphenyls, diphenylalkanes, urea derivatives, oxygen andnitrogen acetals and formals, benzamidines, isothiazolines, phthalimidederivatives, pyridine derivatives, antimicrobial surface-activecompounds, guanidines, antimicrobial amphoteric compounds, quinolines,1,2-dibromo-2,4-dicyanobutane, iodo-2-propylbutyl carbamate, iodine,iodophors, peroxo compounds, halogen compounds and any desired mixturesof the above.

The antimicrobial active compound can be selected here from ethanol,n-propanol, propanol, 1,3-butanediol, phenoxyethanol, 1,2-propyleneglycol, glycerol, undecylenic acid, benzoic acid, salicylic acid,dihydracetic acid, o-phenylphenol, N-methylmorpholineacetonitrile (MMA),2-benzyl-4-chlorophenol, 2,2′-methylenebis(6-bromo-4-chlorophenol),4,4′-dichloro-2′-hydroxydiphenyl ether (dichlosan),2,4,4′-trichloro-2′-hydroxydiphenyl ether (trichlosan), chlorhexidine,N-(4-chlorophenyl)-N-(3,4-dichlorophenyl)urea,N,N′-(1,10-decanediyldi-1-pyridinyl-4-ylidene)bis(1-octanamine)dihydrochloride,N,N′-bis(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetraazatetradecanediimid-amide,glucoprotamines, antimicrobial surface-active quaternary compounds,guanidines including the bi- and poly-guanidines, such as, for example,1,6-bis(2-ethylhexy/biguanidohexane) dihydrochloride,1,6-di(N1,N1′-phenyldiguanido-N5,N5′)-hexane tetrahydrochloride,1,6-di(N1,N1′-phenyl-N,N1-methyldiguanido-N5,N5′)-hexanedihydrochloride, 1,6-di(N1,N1′-o-chlorophenyldiguanido-N5,N5′)-hexanedihydrochloride,1,6-di(N1,N1′-2,6-dichlorophenyldiguanido-N5,N5′)-hexanedihydrochloride,1,6-di(N1,N1′-beta-(p-methoxyphenyl)diguanido-N5,N5′-hexanedihydrochloride,1,6-di(N1,N1′-alpha-methyl-beta-phenyldiguanido-N5,N5′)-hexanedihydrochloride, 1,6-di(N1,N1′-p-nitrophenyldiguanido-N5,N5′)-hexanedihydrochloride,omega:omega-di(N1,N1′-phenyldiguanido-N5,N5′)-di-n-propyl etherdihydrochloride,omega:omega′-di(N1,N1′-p-chlorophenyldiguanido-N5,N5′)-di-n-propyl ethertetrahydrochloride,1,6-di(N1,N1′-2,4-dichlorophenyldiguanido-N5,N5′)-hexanetetrahydrochloride, 1,6-di(N1,N1′-p-methylphenyldiguanido-N5,N5′)-hexanedihydrochloride,1,6-di(N1,N1-2,4,5-trichlorophenyldiguanido-N5,N5′)-hexanetetrahydrochloride,1,6-di[N1,N1′-alpha-(p-chlorophenyl)ethyldiguanido-N5,N5′]-hexanedihydrochloride, omega:omega-di(N1,N1′-p-chlorophenyldiguanido-N5,N5′)-m-xylene dihydrochloride,1,12-di(N1,N1′-p-chlorophenyldiguanido-N5,N5′)-dodecane dihydrochloride,1,10-di(N1,N1′-phenyldiguanido-N5,N5′)-decane tetrahydrochloride,1,12-di(N1,N1′-phenyldiguanido-N5,N5′)-dodecane tetrahydrochloride,1,6-di(N1,N1′-o-chlorophenyldiguanido-N5,N5′)-hexane dihydrochloride,1,6-di(N1,N1′-o-chlorophenyldiguanido-N5,N5′)-hexane tetrahydrochloride,ethylenebis(1-tolylbiguanide), ethylenebis(p-tolylbiguanides),ethylenebis(3,5-dimethylphenylbiguanide),ethylenebis(p-tert-amylphenylbiguanide),ethylenebis(nonylphenylbiguanide), ethylenebis(phenylbiguanide),ethylenebis(N-butylphenylbiguanide),ethylenebis(2,5-diethoxyphenylbiguanide),ethylenebis(2,4-dimethylphenylbiguanide),ethylenebis(o-diphenylbiguanide), ethylenebis(mixed amylnaphthylbiguanide), N-butylethylenebis(phenylbiguanide),trimethylene-bis(o-tolylbiguanide),N-butyltrimethylenebis(phenylbiguanides) and the corresponding saltssuch as acetates, gluconates, hydrochlorides, hydrobromides, citrates,bisulfites, fluorides, polymaleates, N-coconut alkylsarcosinates,phosphites, hypophosphites, perfluorooctanoates, silicates, sorbates,salicylates, maleates, tartrates, fumarates,ethylene-diaminetetraacetates, iminodiacetates, cinnamates,thiocyanates, arginates, pyromellitates, tetracarboxybutyrates,benzoates, glutarates, monofluorophosphates, perfluoropropionates andany desired mixtures thereof. Furthermore, halogenated xylene and cresolderivatives, such as p-chlorometacresol or p-chlorometaxylene, andnatural antimicrobial active compounds of plant origin (for example fromspices or herbs), of animal and of microbial origin are suitable.Preferably, surface-active quaternary compounds having antimicrobialactivity, a natural antimicrobial active compound of plant origin and/ora natural antimicrobial active compound of animal origin, extremelypreferably at least one natural antimicrobial active compound of plantorigin from the group comprising caffeine, theobromine and theophylline,and essential oils such as eugenol, thymol and geraniol, and/or at leastone natural antimicrobial active compound of animal origin from thegroup comprising enzymes such as albumin from milk, lysozyme andlactoperoxidase, and/or at least one surface-active quaternary compoundhaving antimicrobial activity containing an ammonium, sulfonium,phosphonium, iodonium or arsonium group, peroxo compounds and chlorocompounds can be employed. Substances of microbial origin,“bacteriocins”, can also be employed.

The quaternary ammonium compounds (QAC) suitable as antimicrobial activecompounds have the general formula (R¹)(R²)(R³)(R⁴)N+X—, in which R¹ toR⁴ are identical or different C₁-C₂₂-alkyl radicals, C₇-C₂₈-aralkylradicals or heterocyclic radicals, where two or, in the case of anaromatic integration as in pyridine, even three radicals together withthe nitrogen atom form the heterocycle, for example a pyridinium orimidazolinium compound, and X— are halide ions, sulfate ions, hydroxideions or similar anions. For an optimum antimicrobial action, preferablyat least one of the radicals has a chain length of 8 to 18, inparticular, 12 to 16 C atoms.

QAC can be prepared by reaction of tertiary amines with alkylatingagents, such as, for example, methyl chloride, benzyl chloride, dimethylsulfate, dodecyl bromide, but also ethylene oxide. The alkylation oftertiary amines having a long alkyl radical and two methyl groups isparticularly easily possible, and the quaternization of tertiary amineshaving two long radicals and a methyl group can be carried out undermild conditions with the aid of methyl chloride. Amines which have threelong alkyl radicals or hydroxy-substituted alkyl radicals are lessreactive and are preferably quaternized using dimethyl sulfate.

Suitable QAC are, for example, benzalkonium chloride(N-alkyl-N,N-dimethylbenzylammonium chloride, CAS No. 8001-54-5),Benzalkon B (m,p-dichlorobenzyldimethyl-C₁₋₂-alkylammonium chloride, CASNo. 58390-78-6), benzoxonium chloride(benzyldodecyl-bis(2-hydroxyethyl)ammonium chloride), cetrimoniumbromide (N-hexadecyl-N,N-trimethylammonium bromide, CAS No. 57-09-0),benzetonium chloride(N,N-dimethyl-N-[2-[2-[p-(1,1,3,3-tetramethylbutyl)phenoxy]ethox-y]ethyl]-benzylammoniumchloride, CAS No. 121-54-0), dialkyldimethylammonium chlorides such asdi-n-decyldimethylammonium chloride (CAS No. 7173-51-5-5),didecyldimethylammonium bromide (CAS No. 2390-68-3),dioctyldimethylammoniumchloric, 1-cetylpyridinium chloride (CAS No.123-03-5) and thiazoline iodide (CAS No. 15764-48-1) and their mixtures.Particularly preferred QAC are the benzalkonium chlorides havingC8-C18-alkyl radicals, in particular C12-C14-alkylbenzyldimethylammoniumchloride.

Benzalkonium halides and/or substituted benzalkonium halides are, forexample, commercially obtainable as Barquat® ex Lonza, Marquat® exMason, Variquat® ex Witco/Sherex and Hyamine® ex Lonza, and Bardac® exLonza. Further commercially obtainable antimicrobial active compoundsare N-(3-chloroallyl)hexaminium chloride such as Dowicide® and Dowicil®ex Dow, benzethonium chloride such as Hyamine® 1622 ex Rohm & Haas,methylbenzethonium chloride such as Hyamine® 10× ex Rohm & Haas,cetylpyridinium chloride such as cepacol chloride ex Merrell Labs.

The antimicrobial active compounds are employed in amounts of 0.0001% byweight to 1% by weight, preferably of 0.001% by weight to 0.8% byweight, particularly preferably of 0.005% by weight to 0.3% by weightand in particular of 0.01 to 0.2% by weight.

The detergents or cleaners according to the invention can contain UVabsorbents (UV absorbers), which become attached to the treated textilesand improve the light resistance of the fibers and/or the lightresistance of other recipe constituents. UV absorbers are to beunderstood as meaning organic substances (lightscreen filters) which areable to absorb ultraviolet rays and emit the absorbed energy again inthe form of longer-wave radiation, for example heat.

Compounds which have these desired properties are, for example, thecompounds and derivatives of benzophenone having substituents in the 2-and/or 4-position which are active by radiationless deactivation.Furthermore, substituted benzotriazoles, acrylates phenyl-substituted inthe 3-position (cinnamic acid derivatives, optionally having cyanogroups in the 2-position), salicylates, organic Ni complexes and naturalsubstances such as umbelliferone and the endogenous urocanic acid arealso suitable. Biphenyl and especially stilbene derivatives such as aredescribed, for example, in EP 0728749 A and are commercially obtainableas Tinosorb® FD or Tinosorb® FR ex Ciba have particular importance. Thefollowing may be mentioned as UV-B absorbers: 3-benzylidenecamphor or3-benzylidenenorcamphor and its derivatives, for example3-(4-methylbenzy-lidene)camphor, such as described in EP 0693471 B1;4-aminobenzoic acid derivatives, preferably 2-ethylhexyl4-(dimethylamino)benzoate, 2-octyl 4-(dimethylamino)benzoate and amyl4-(dimethylamino)benzoate; esters of cinnamic acid, preferably2-ethylhexyl 4-methoxycinnamate, propyl 4-methoxycinnamate, isoamyl4-methoxycinnamate, 2-ethylhexyl 2-cyano-3,3-phenylcinnamate(octocrylene); esters of salicylic acid, preferably 2-ethylhexylsalicylate, 4-isopropylbenzyl salicylate, homomethyl salicylate;derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone,2-hydroxy-4-methoxy-4′-methylbenzophenone,2,2′-dihydroxy-4-methoxybenzophenone; esters of benzalmalonic acid,preferably di-2-ethylhexyl 4-methoxybenzmalonate; triazine derivatives,such as, for example,2,4,6-trianilino-(p-carbo-2′-ethyl-1′-hexyloxy)-1,3,5-triazine and octyltriazone, such as described in EP 0818450 A1 or dioctyl butamidotriazone (Uvasorb® HEB); propane-1,3-diones, such as, for example,1-(4-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione;ketotricyclo(5.2.1.0)decane derivatives, such as described in EP 0694521B1. 2-phenylbenzimidazole-5-sulfonic acid and its alkali metal, alkalineearth metal, ammonium, alkylammonium, alkanolammonium and glucammoniumsalts are further suitable; sulfonic acid derivatives of benzophenones,preferably 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid and itssalts; sulfonic acid derivatives of 3-benzylidenecamphor, such as, forexample, 4-(2-oxo-3-bornylidenemethyl)benzenesulfonic acid and2-methyl-5-(2-oxo-3-bornylidene)sulfonic acid and their salts.

Suitable typical UV-A filters are, in particular, derivatives ofbenzoylmethane, such as, for example,1-(4′-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione,4-tert-butyl-4′-methoxydibenzoyl-methane (Parsol 1789),1-phenyl-3-(4′-isopropylphenyl)propane-1,3-dione and enamine compounds,such as described in DE 19712033 A1 (BASF). The UV-A and UV-B filterscan of course also be employed as mixtures. In addition to the solublesubstances mentioned, insoluble lightscreen pigments, namely finelydisperse, preferably nanoized metal oxides or salts are also suitablefor this purpose. Examples of suitable metal oxides are in particularzinc oxide and titanium dioxide and oxides of iron, zirconium, silicon,manganese, aluminum and cerium and their mixtures. As salts, silicates(talc), barium sulfate or zinc stearate can be employed. The oxides andsalts are already used in the form of the pigments for skin-caring andskin-protecting emulsions and decorative cosmetics. The particles shouldin this case have a mean diameter of less than 100 nm, preferablybetween 5 and 50 nm and in particular between 15 and 30 nm. They canhave a spherical shape, but also those particles can be used which havean ellipsoidal shape or a shape differing in other ways from thespherical shape. The pigments can also be present in surface-treated,that is hydrophilicized or hydrophobicized, form. Typical examples arecoated titanium dioxides, such as, for example, titanium dioxide T 805(Degussa) or Eusolex® T2000 (Merck; suitable hydrophobic coating agentsfor this are preferably silicones and particularly preferablytrialkoxyoctylsilanes or simethicones. Preferably, micronized zinc oxideis used. Further suitable UV lightscreen filters can be taken from thesurvey of P. Finkel in the SÖFW-Journal 122 (1996), p. 543.

The UV absorbents are customarily employed in amounts from 0.01% byweight to 5% by weight, preferably from 0.03% by weight to 1% by weight.

For increasing the washing or cleaning performance, compositionsaccording to the invention can contain further enzymes, where inprinciple all enzymes established for these purposes in the prior artcan be employed. These include, in particular, further proteases,amylases, lipases, hemicellulases, cellulases or oxidoreductases, andpreferably their mixtures. These enzymes are in principle of naturalorigin; starting from the natural molecules, for use in detergents andcleaners improved variants are available, which are correspondinglypreferably employed. Compositions according to the invention containenzymes preferably in total amounts of 1×10-8 to 5 percent by weightbased on active protein.

Among the further proteases, those of the subtilisin type are preferred.Examples of these are the subtilisins BPN′ and Carlsberg, the proteasePB92, the subtilisins 147 and 309, the alkaline protease from Bacilluslentus, subtilisin DY and the enzymes to be assigned thermitase,proteinase K and the proteases TW3 and TW7 to the subtilases, but nolonger to the subtilisins in the narrower sense. Subtilisin Carlsberg isavailable in further developed form under the trade name Alcalase® fromNovozymes A/S, Bagsvaerd, Denmark. The subtilisins 147 and 309 aremarketed under the trade names Esperase®, or Savinase® by Novozymes.From the protease from Bacillus lentus DSM 5483 (WO 91/02792 A1) arederived the variants routed under the designation BLAP®, which aredescribed in particular in WO 92/21760 A1, WO 95/23221 A1, WO 021088340A2 and WO 03/038082 A2. Further usable proteases from various Bacillussp. and B. gibsonii follow from the patent applications WO 03/054185 A1,WO 03/056017 A2, WO 03/055974 A2 and WO 03/054184 A1, already mentionedintroductorily.

Further usable proteases are, for example, the enzymes obtainable underthe trade names Durazym®, Relase®, Everlase®, Nafizym, Natalase®,Kannase® and Ovozymes® from Novozymes, the enzymes obtainable under thetrade names Purafect®, Purafect® OxP and Properase® from Genencor, theenzymes obtainable under the trade name Protosol® from AdvancedBiochemicals Ltd., Thane, India, the enzymes obtainable under the tradename Wuxi® from Wuxi Snyder Bioproducts Ltd., China, the enzymesobtainable under the trade names Proleather® and Protease P® from AmanoPharmaceuticals Ltd., Nagoya, Japan, and the enzyme obtainable under thename Proteinase K-16 from Kao Corp., Tokyo, Japan.

Examples of amylases employable according to the invention are the.alpha.-amylases from Bacillus licheniformis, from B. amyloliquefaciensor from B. stearothermophilus and their improved further developmentsfor use in detergents and cleaners. The enzyme from B. licheniformis isobtainable from Novozymes under the name Termamyl® and from Genencorunder the name Purastar®ST.

Further development products of this α-amylase are obtainable fromNovozymes under the trade names Duramyl® and Termamyl®ultra, fromGenencor under the name Purastar®OxAm and from Daiwa Seiko Inc., Tokyo,Japan, as Keistase®. The α-amylase of B. amyloliquefaciens is marketedby Novozymes under the name BAN®, and derived variants of the.alpha.-amylase from B. stearothermophilus under the names BSG® andNovamyl®, likewise from Novozymes.

In addition, for this purpose the α-amylase from Bacillus sp. A 7-7 (DSM12368) disclosed in the application WO 02/10356 A2 and the cyclodextringlucanotransferase (CGTase) from B. agaradherens (DSM 9948) described inthe application WO 02/44350 A2 are to be stressed. Furthermore, theamylolytic enzymes can be employed which are part of the sequence spaceof α-amylases, which is defined in the application WO 03/002711 A2, andthose which are defined in the application WO 03/054177 A2. Likewise,fusion products of said molecules can be employed, for example, thosefrom the application DE 10138753 A1.

Moreover, the further developments of the α-amylase from Aspergillusniger and A. oryzae obtainable under the trade name Fungamyl® fromNovozymes are suitable. A further commercial product is, for example,Amylase-LT®.

Compositions according to the invention can contain lipases orcutinases, in particular because of their triglyceride-cleavingactivities, but also in order to produce peracids in situ from suitableprecursors. These include, for example, the lipases originallyobtainable from Humicola lanuginosa (Thermomyces lanuginosus), orfurther developed, in particular those having the amino acid replacementD96L. They are marketed, for example, by Novozymes under the trade namesLipolase®, Lipolase®Ultra, LipoPrime®, Lipozyme® and Lipex®. Inaddition, for example, the cutinases can be employed which haveoriginally been isolated from Fusarium solani pisi and Humicolainsolens. Lipases likewise usable are obtainable from Amano under thenames Lipase CE®, Lipase P®, Lipase B®, or Lipase CES®, Lipase AKG®,Bacillis sp. Lipase®, Lipase AP®, Lipase M-AP® and Lipase AML®. FromGenencor, for example, the lipases, or cutinases can be employed whosestarting enzymes have originally been isolated from Pseudomonasmendocina and Fusarium solanii. Further important commercial productswhich may be mentioned are the preparations M1 Lipase® and Lipomax®originally marketed by Gist-Brocades and the enzymes marketed by MeitoSangyo KK, Japan, under the names Lipase MY-30®, Lipase OF® and LipasePL®, furthermore the product Lumafast® from Genencor.

Compositions according to the invention, in particular if they areintended for the treatment of textiles, can contain cellulases,depending on the purpose as pure enzymes, as enzyme preparations or inthe form of mixtures, in which the individual components advantageouslycomplement each other with respect to their various performance aspects.These performance aspects include, in particular, contributions to theprimary washing performance, to the secondary washing performance of thecomposition (antiredeposition action or graying inhibition) and reviving(fabric action), to the exertion of a “stone washed” effect.

A usable fungal, endoglucanase (EG)-rich cellulase preparation, or itsfurther developments are supplied by Novozymes under the trade nameCelluzyme®. The products Endolase® and Carezyme® likewise obtainablefrom Novozymes are based on the 50 kD EG, or the 43 kD EG, from H.insolens DSM 1800. Further commercial products from this company whichcan be employed are Cellusoft® and Renozyme®. The latter is based on theapplication WO 96/29397 A1. Improved-performance cellulase variantsfollow, for example, from the application WO 98/12307 A1. Likewise, thecellulases disclosed in the application WO 97/14804 A1 can be employed;for example the 20 kD EG from Melanocarpus disclosed therein, which isobtainable from AB Enzymes, Finland, under the trade names Ecostone® andBiotouch®. Further commercial products from AB Enzymes are Econase® andEcopulp®. Further suitable cellulases from Bacillus sp. CBS 670.93 andCBS 669.93 are disclosed in WO 96/34092 A2, where the CBS 670.93 fromBacillus sp. is obtainable from Genencor under the trade name Puradax®.Further commercial products from Genencor are “Genencor detergentcellulase L” and IndiAge®Neutra.

Compositions according to the invention can contain, in particular forthe removal of certain problem soiling, further enzymes which aresummarized under the term hemicellulases. These include, for examplemannanases, xanthanlyases, pectinlyases (=pectinases), pectin esterases,pectate lyases, xyloglucanases (=xylanases), pullulanases andβ-glucanases. Suitable mannanases are obtainable, for example, under thenames Gamanase® and Pektinex AR® from Novozymes, under the name Rohapec®B1 L from AB Enzymes and under the name Pyrolase® from Diversa Corp.,San Diego, Calif., USA. A suitable β-glucanase from a B. alcalophilusfollows, for example, from the application WO 99/06573 A1. Theβ-glucanase prepared from B. subtilis is obtainable under the nameCereflo® from Novozymes.

For increasing the bleaching action, detergents and cleaners accordingto the invention can contain oxidoreductases, for example oxidases,oxygenases, catalases, peroxidases, such as halo-, chloro-, bromo-,lignin, glucose or manganese peroxidases, dioxygenases or laccases(phenol oxidases, polyphenol oxidases). Suitable commercial productswhich may be mentioned are Denilite® 1 and 2 from Novozymes.Advantageously, preferably organic, particularly preferably aromaticcompounds interacting with the enzymes are additionally added in orderto increase the activity of the oxidoreductases concerned (enhancers) orin order, in the case of greatly different redox potentials between theoxidizing enzymes and the soiling to guarantee the electron flux(mediators).

The enzymes employed in compositions according to the invention areeither derived originally from microorganisms, for example of the generaBacillus, Streptomyces, Humicola, or Pseudomonas, and/or produced bysuitable microorganisms by bio-technological methods known per se, forexample by transgenic expression hosts of the genera Bacillus orfilamentous fungi.

The purification of the enzymes concerned is more favorably carried outby methods established per se, for example by means of precipitation,sedimentation, concentration, filtration of the liquid phases,microfiltration, ultrafiltration, action of chemicals, deodorization orsuitable combinations of these steps.

The enzymes can be added to compositions according to the invention inany form established according to the prior art. These include, forexample, the solid preparations obtained by granulation, extrusion orlyophilization or, in particular in the case of liquid or gelatinouscompositions, solutions of the enzymes, advantageously as concentratedas possible, low in water and/or treated with stabilizers.

Alternatively, the enzymes can be encapsulated both for the solid andfor the liquid administration form, for example by spray drying orextrusion of the enzyme solution together with a, preferably natural,polymer or in the form of capsules, for example those in which theenzymes are enclosed as in a solidified gel or in those of thecore-shell type, in which an enzyme-containing core is covered with awater-, air- and/or chemical-impermeable protective layer. Insuperimposed layers, further active compounds, for example stabilizers,emulsifiers, pigments, bleaches or colorants, can be applied. Capsulesof this type are applied by methods known per se, for example by shakeror roller granulation or in fluidized-bed processes. Advantageously,granules of this type are low in dust, for example as a result ofapplying polymeric film-forming agents, and storage-stable on account ofthe coating.

Furthermore, it is possible to package two or more enzymes together,such that individual granules have a number of enzyme activities.

A protein and/or enzyme contained in a composition according to theinvention can be protected, particularly during storage, against damagesuch as, for example, inactivation, denaturation or disintegration, forexample by means of physical influences, oxidation or proteolyticcleavage. In the case of microbial preparation of the proteins and/orenzymes, an inhibition of the proteolysis is particularly preferred, inparticular if the compositions also contain proteases. Preferredcompositions according to the invention for this purpose containstabilizers.

One group of stabilizers are reversible protease inhibitors. Frequently,for this purpose, benzamidine hydrochloride, borax, boric acids, boronicacids or their salts or esters are employed, among them especiallyderivatives having aromatic groups, for example ortho-, meta- orpara-substituted phenylboronic acids, in particular4-formylphenylboronic acid, or the salts or esters of said compounds.Peptide aldehydes, that is, oligopeptides having a reduced C-terminus,in particular, those consisting of 2 to 50 monomers, are also employedfor this purpose. The peptidic reversible protease inhibitors include,inter alia, ovomucoid and leupeptin. Specific, reversible peptideinhibitors for the protease subtilisin and fusion proteins of proteasesand specific peptide inhibitors are also suitable for this.

Further enzyme stabilizers are amino alcohols such as mono-, di-,triethanol- and -propanolamine and their mixtures, aliphatic carboxylicacids up to C12, such as, for example, succinic acid, other dicarboxylicacids or salts of said acids. End group-closed fatty acid amidealkoxylates are also suitable for this purpose. Certain organic acidsemployed as builders are able, as disclosed in WO 97/18287, toadditionally stabilize an enzyme present.

Lower aliphatic alcohols, but especially polyols, such as, for example,glycerol, ethylene glycol, propylene glycol or sorbitol are furtherfrequently employed enzyme stabilizers. Diglycerol phosphate alsoprotects against denaturation by physical influences. Likewise, calciumand/or magnesium salts are employed, such as for example calcium acetateor calcium formate.

Polyamide oligomers or polymeric compounds such as lignin, water-solublevinyl copolymers or cellulose ethers, acrylic polymers and/or polyamidesstabilize the enzyme preparation, inter alia, to physical influences orpH variations. Polyamine N-oxide-containing polymers simultaneously actas enzyme stabilizers and as color transfer inhibitors. Other polymericstabilizers are linear C₈-C₁₈ polyoxyalkylenes. Alkylpolyglycosides canalso stabilize the enzymatic components of the composition according tothe invention and are preferably able additionally to increase these intheir performance. Crosslinked N-containing compounds preferably fulfilla dual function as soil release agents and as enzyme stabilizers.Hydrophobic, nonionic polymer in particular stabilizes an optionallycontained cellulase.

Reductants and antioxidants increase the stability of the enzymes tooxidative disintegration; sulfur-containing reductants, for example, arecommon for this. Other examples are sodium sulfite and reducing sugar.

Combinations of stabilizers, for example of polyols, boric acid and/orborax, the combination of boric acid or borate, reducing salts andsuccinic acid or other dicarboxylic acids or the combination of boricacid or borate with polyols or polyamino compounds and with reducingsalts are particularly preferably employed. The action of peptidealdehyde stabilizers is more favorably increased by the combination withboric acid and/or boric acid derivatives and polyols and still furtherby the additional action of divalent cations, such as, for example,calcium ions.

Since compositions according to the invention can be supplied in allconceivable forms, enzymes according to the invention, or proteins inall formulations expedient for the addition to the respectivecompositions, are respective embodiments of the present invention. Theseinclude, for example, liquid formulations, solid granules or capsules.

The encapsulated form is suggested in order to protect the enzymes orother ingredients from other constituents, such as, for example,bleaches, or in order to make possible a controlled release. Dependingon the size of these capsules, a distinction is made according tomilli-, micro- and nanocapsules, microcapsules being particularlypreferred for enzymes. Such capsules are disclosed, for example, by thepatent applications WO 97/24177 and DE 19918267. A possibleencapsulation method consists in encapsulating the proteins, startingfrom a mixture of the protein solution with a solution or suspension ofstarch or a starch derivative, in this substance. Such an encapsulationprocess is described by the application WO 01/38471.

In the case of solid compositions, the proteins can be employed, forexample, in dried, granulated and/or encapsulated form. They can beadded separately, that is, as an individual phase, or with otherconstituents together in the same phase, with or without compaction. Ifmicroencapsulated enzymes are to be processed in solid form, the watercan be removed from the aqueous solutions resulting from the workupusing processes known from the prior art, such as spray drying,centrifuging or by resolubilizing. The particles obtained in this waycustomarily have a particle size between 50 and 200 μm.

The enzymes, and also the protein according to the invention startingfrom a protein obtainment and preparation carried out according to theprior art, can be added to liquid, gelatinous or pasty compositionsaccording to the invention in concentrated aqueous or nonaqueoussolution, suspension or emulsion, but also in gel form or encapsulatedor as a dried powder.

Detergents or cleaners according to the invention of this type are as arule prepared by simple mixing of the ingredients, which can be added toan automatic mixer in substance or as a solution.

In addition to the primary washing performance, the proteases containedin detergents can further fulfill the function of activating otherenzymatic constituents by proteolytic cleavage or inactivating after anappropriate time of action, i.e., as has been disclosed, for example, inapplications WO 94/29426 or EP 747471. Comparable regulatory functionsare also possible by means of the protein according to the invention.One embodiment of the present invention is furthermore thosecompositions containing capsules of protease-sensitive material, which,for example, are hydrolyzed by proteins according to the invention at anintended point in time and release their contents. A comparable effectcan also be achieved with other multiphase compositions.

A further embodiment is compositions for the treatment of textile rawmaterials or for textile care which contain an alkaline proteaseaccording to the invention.

A further embodiment is compositions for the treatment of fibers ortextiles containing natural constituents, in particular of thosecontaining wool or silk.

Natural fibers in particular, such as wool or silk, are distinguished bya characteristic, microscopic surface structure. This can lead long-termto undesired effects, as has been explained in the example of wool inthe article by R. Breier in Melliand's Textilberichte [Melliand'sTextile Reports] of 4.1.2000 (p. 263), such as felting. To avoid sucheffects, the natural raw materials are treated with compositionsaccording to the invention, which, for example, contribute to smoothingthe scaly surface structure based on protein structures and thuscounteract felting.

In a preferred embodiment, the composition is conceived using a proteaseaccording to the invention such that it can be used regularly as a carecomposition, for example by adding it to the washing process, using itafter washing or applying it independently of washing. The desiredeffect consists in obtaining a smooth surface structure of the textileover a long period of time and/or preventing and/or reducing damage tothe fabric.

A separate subject of the invention is methods for the mechanicalcleaning of textiles or of hard surfaces, in which an alkaline proteaseaccording to the invention becomes active at least in one of the methodsteps.

Among these, those methods are preferred in which the alkaline proteaseaccording to the invention is employed in an amount from 40 μg to 4 g,preferably from 50 μg to 3 g, particularly preferably from 100 μg to 2 gand very particularly preferably from 200 μg to 1 g per application.Included are all integral and nonintegral values in each case lyingbetween these numbers.

Under this fall both manual and mechanical methods, mechanical methodsbeing preferred on account of their more precise controllability, asfar, for example, as the amounts employed and times of action areconcerned.

Methods for the cleaning of textiles are in general distinguished inthat in a number of method steps various cleaning-active substances areapplied to the article to be cleaned and, after the time of action, arewashed off, or in that the article to be cleaned is treated in anotherway with a detergent or a solution of this composition. The same appliesfor methods for the cleaning of all other materials than textiles whichare summarized under the term hard surfaces. All conceivable washing orcleaning methods can be enriched in at least one of the method steps byproteins according to the invention, and are then embodiments of thepresent invention.

Since preferred enzymes according to the invention naturally alreadyhave a protein-dissolving activity and also display this in media whichotherwise have no cleaning power, such as, for example, in mere buffer,an individual substep of such a method for the mechanical cleaning oftextiles can consist in applying an enzyme according to the invention asthe only cleaning-active component, if desired, in addition tostabilizing compounds, salts or buffer substances. This is aparticularly preferred embodiment of the present invention.

In a further preferred embodiment of such methods, the alkaline proteaseaccording to the invention concerned is prepared within the scope of oneof the abovementioned recipes for compositions according to theinvention, preferably detergents, or cleaners, according to theinvention.

Preferred embodiments of this subject of the invention are methods forthe treatment of textile raw materials or for textile care, in which inat least one of the method steps an alkaline protease according to theinvention becomes active.

Among these, methods for textile raw materials, fibers or textilescontaining natural constituents are preferred, and very particularly forthose containing wool or silk.

In this case, for example, this can be methods in which materials forprocessing to textiles are prepared, for example for antifelt finishing,or, for example, methods which enrich the cleaning of worn textiles by acaring component. Because of the action described above of proteases onnatural, protein-containing raw materials, in preferred embodimentsthese are methods for the treatment of textile raw materials, fibers ortextiles containing natural constituents, in particular containing woolor silk.

An individual subject of the invention is the use of an alkalineprotease according to the invention described above for the cleaning oftextiles or of hard surfaces.

Accordingly, the abovementioned concentration ranges apply for theseuses.

Proteases according to the invention can be used, in particularaccording to the properties described above and to the methods describedabove in order to eliminate protein-containing impurities from textilesor from hard surfaces. Embodiments are, for example, the hand wash, themanual removal of spots from textiles or from hard surfaces or the usein connection with a mechanical method.

In a preferred embodiment of this use, the alkaline proteases accordingto the invention concerned are prepared in the scope of one of theabovementioned recipes for compositions according to the invention,preferably detergents, or cleaners.

A further embodiment of this subject of the invention is the use of analkaline protease according to the invention for the activation ordeactivation of ingredients of detergents or cleaners.

As is known, protein constituents of detergents or cleaners can beinactivated by the action of a protease. To employ this otherwise ratherundesired effect is a subject of the present invention. Likewise, it ispossible as described above that by proteolysis, another component isfirst activated, for example if it is a hybrid protein of the actualenzyme and the inhibitor appropriate therefor, as has been disclosed,for example, in the application WO 00/01831 A2. Another example of sucha regulation is that in which an active component, for the protection orfor the control of its activity, is present encapsulated in a materialwhich is attacked by proteolysis. Proteins according to the inventioncan thus be used for inactivation, activation or release reactions, inparticular in multiphase compositions.

Corresponding to what has been said above, the following uses are alsoembodiments of the present invention:

-   -   The use of an alkaline protease according to the invention for        the preparation or treatment of raw materials or intermediates        in textile production, in particular for the removal of        protective layers on fabrics;    -   the use of an alkaline protease according to the invention for        the treatment of textile raw materials or for textile care and        among these preferably    -   the corresponding use for textile raw materials, fibers or        textiles containing natural constituents and very particularly        for those containing wool or silk.

The present invention is also realized in the form of those compositionscomprising an alkaline protease according to the invention, which arecosmetics. Among these, all types of cleaning and caring compositionsfor human skin or hair are understood, in particular cleaningcompositions.

Proteases also play a crucial role in the cell renewal process of thehuman skin (desquamation) (T. Egelrud et al., Acta Derm. Venerol., Vol.71 (1991), pp. 471-474). Accordingly, proteases are also used asbioactive components in skincare compositions in order to assist thebreakdown of the desmosome structures which are increased in dry skin.The use of subtilisin proteases with amino acid replacements inpositions R99GA/S, S154D/E and/or L211D/E for cosmetic purposes isdescribed, for example, in WO 97/07770 A1. Corresponding to what hasbeen said above, proteases according to the invention can be furtherdeveloped by means of the corresponding point mutations. Thus proteasesaccording to the invention are also suitable, in particular those which,for example, after mutagenesis or by addition of correspondingsubstances interacting with them, are controlled in their activity, asactive components in skin- or hair-cleaning or care compositions. Thosepreparations of these enzymes are particularly preferred which, asdescribed above, for example by coupling to macromolecular carriers(compare U.S. Pat. No. 5,230,891) are stabilized and/or derivatized bypoint mutations in highly allergenic positions such that they have ahigher skin compatibility for humans.

Accordingly, corresponding cosmetic cleaning and care methods and theuse of proteolytic enzymes of this type for cosmetic purposes are alsoincluded in this subject of the invention, in particular incorresponding compositions, such as, for example, shampoos, soaps orwashing lotions, or in care compositions which are supplied, forexample, in the form of creams. The use in a peeling pharmaceutical, orfor its production, is also included in this claim.

In addition to the use in detergents and cleaners and cosmetics,numerous possibilities of application of proteases, in particularsubtilases, are established in the prior art. The handbook Industrialenyzmes and their applications by H. Uhlig, Wiley-Verlag, New York(1998) presents an overview of this. All these techniques can beenriched by alkaline proteases according to the invention. Should itemerge that they can be further developed by the use of proteasesaccording to the invention, these are included in the scope ofprotection of the present invention. This includes, in particular, thefollowing areas of use:

-   -   the use of an alkaline protease according to the invention for        the biochemical analysis or for the synthesis of low molecular        weight compounds or of proteins;    -   among these preferably the use for the end group determination        in the scope of a peptide sequence analysis;    -   the use of an alkaline protease according to the invention for        the preparation, purification or synthesis of natural substances        or biological valuable substances, preferably within the scope        of corresponding compositions or methods;    -   the use of an alkaline protease according to the invention for        the synthesis of proteins or other low molecular weight chemical        compounds;    -   the use of an alkaline protease according to the invention for        the treatment of natural raw materials, in particular for        surface treatment, very particularly in a method for the        treatment of leather, preferably within the scope of        corresponding compositions or methods;    -   the use of an alkaline protease according to the invention for        the treatment of photographic films, in particular for the        removal of gelatin-containing or similar protective layers; and    -   the use of an alkaline protease according to the invention for        the preparation of foods or of feeds.

In principle, is comprised in the present application any technicalfield, which is enriched by the novel alkaline proteases presentedhereby or methods or uses based hereon.

The following examples illustrate the present invention withoutrestricting it thereto.

EXAMPLES

All molecular biological working steps follow standard methods, such asare indicated, for example, in the handbook by Fritsch, Sambrook andManiatis, Molecular cloning: a laboratory manual, Cold Spring HarbourLaboratory Press, New York (1989), or comparable relevant works. Enzymesand kits were employed according to the instructions of the respectivemanufacturers.

Example 1 Obtainment of Cell Material from Soil Habitats

Soil samples were taken from various locations in Germany, taken up inwater and suspended substances were sedimented by allowing to stand for30 minutes. The supernatant was plated out on 5% strength agar platesusing HSP10 solid medium (0.1 g of yeast extract, Difco, Heidelberg; 0.1g of casein peptone, tryptically digested, Difco; 0.1 g of solublestarch (Merck, Order No. 1.01251); 2 g of Na₂CO₃; to 1000 ml ofdistilled water; pH10) and cultured at 30° C. for about 2 weeks. Thebacterial lawn obtained was recovered mechanically from the agarsurface.

Example 2 Arrangement of an Expression Gene Bank

The expression system chosen was the vector pUC18 (GenBank, NationalInstitutes of Health, Bethesda, Md., USA; accession number L08752; FIG.3) in Escherichia coli DH12S. This vector carries the β-galactosidasepromoter of the lac operon inducible by addition of IPTG, so that inthese cells a hereby controlled expression of the DNA integrated in themultiple cloning site is possible. The strain DH12S is suitable becauseof its laclq genotype for the IPTG induction and advantageous for aprotease activity screening, because it has a sufficiently lowendogenous proteolytic activity. Preliminary experiments had shown thatE. coli JM109 also fulfills the same requirements.

The workup of the DNA from the sample obtained according to example 1was carried out according to Zhou et al., Appl. Environ. Microbiol.,Vol. 62 (1996), pp. 316-322. This purified metagenomic DNA (see below)was subjected to a preparative partial restriction using the restrictionenzyme Alu I for the preparation of fragment sizes in the range from 2to 10 kb.

For this, first the optimum restriction incubation period was determinedby recording enzyme kinetics. For this, 2.8 μg of the DNA preparationwere incubated at 37° C. in the appropriate reaction buffer supplied bythe manufacturer of Alu I (New England Biolabs, Schwalbach, Germany;Catalog No. R0137S). By addition of 0.2 U of Alu I per μg of DNA, thereaction was started in a total volume of 21 μl and then in two-minuteintervals in each case 1.5 μl were taken from the batch, in which ineach case the reaction was ended immediately by addition of 10 mMTris/HCl, pH 7.0; 20% glycerol; 0.1% SDS and cooling to 0° C. Bysubsequent analysis on a 0.7% strength agarose gel, the optimumrestriction period for a partial digestion was determined. For theisolation of the DNA isolated according to Example 1, it is about 6 to 7minutes in order to obtain fragments in the size range from 2 to 10 kb.

The preparative partial digestion was accordingly carried out in 30parallel batches. After appropriate stopping of the reaction, the batchwas electrophoretically separated on a preparative 0.7% strength agarosegel, the gel region containing DNA of the sizes 2 to 10 kb was excizedand this was isolated by means of electroelution in dialysis tubing at4° C. The DNA was finally precipitated with 1/10 volume of 3 M Naacetate and the 2.5-fold volume of ethanol and taken up in an adequatevolume. For the further separation of any possible present smaller DNAfragments, the gel electrophoresis, electroporation and precipitationwere repeated.

440 ng of the fragmented metagenomic DNA thus obtained were ligatedovernight at 16° C. in a total volume of 15 μl with 150 ng of the vectorpUC18 with addition of 400 NEB units of T4-DNA ligase in 1× ligasebuffer. This vector had been linearized beforehand with Sma I anddephosphorylated with alkaline phosphatase from calf thymus.

The transformation of competent E. coli DH12S cells (Gibco LifeTechnologies, Karlsruhe, catalog number 18312017) was carried out bymeans of electro-transformation. For this, 1 μl of ligation batch and 25μl of cells were mixed, incubated on ice in an electroporation cuvettefor 1 min and treated in the electroporator (BTX® ECM630, GenetronicsInc. San Diego, Calif., USA) according to the manufacturer'sinstructions. After immediate transfer to 1 ml of SOC medium (2%Bacto-tryptone; 0.5% yeast extract; 10 mM NaCl; 2.5 mM KCl; pH 7.0,adjusted with NaOH; autoclaved; supplemented with 10 mM MgSO₄ and MgCl₂and 20 mM D(+)glucose), a recovery phase of 1 h at 37° C. and as inExample 1 plating onto agar plates with HSP10 solid medium followed.

Example 3 Screening for Proteolytic Activity

For the investigation of the quality of the gene bank prepared accordingto Example 2 in E. coli DH12S, the number of primary transformantsproduced altogether and the number of insert-carrying clones wasdetermined by means of blue/white selection in a test plating. For this,1 and 10 μl each of the transformation batch were plated out on 5%strength agar plates with LB medium (10 g of tryptone, 5 g of yeastextract, 5 g of NaCl, 1 ml of 1 N NaOH per 1), which was additionallytreated with 100 μg/ml ampicillin, 0.2 mM (or 4 μg/ml) IPTG and 0.2 mM(or 1 μg/ml) X-Gal, and incubated overnight at 37° C. From 10 whitecolonies, that is, transformants, the plasmids were isolated by means ofminipreparation (kit from Qiagen, Hilden, Germany), a restrictiondigestion was carried out using the restriction enzymes Sac I and HindIII for the excision of the insert (compare FIG. 3) and the fragmentswere separated on a 0.7% strength agarose gel. In fact, all vectorscontained inserts of about 2 to 10 kb size.

The screening of the gene bank produced according to Example 2 wascarried out on 5% strength agar plates 14 cm in diameter using LB mediumampicillin/IPTG/X-Gal (see above) and additionally 2% skimmed milkpowder (Skim Milk, Difco, Order No. 232100). On 10 of these selectionagar plates, corresponding to the titer of the bank volumes of thetransformation batch of in each case about 10 000 cfu were uniformlyplated out by means of glass beads (primary plating).

After incubation at 37° C. for 16 hours, the plates were incubated forup to two weeks at 28° C. During this time, protease-forming clonesrevealed themselves by means of clarification halos in the turbidsubstrate. A separate cell lysis for the detection of nonexportedproteases was not necessary. The validation of the plasmid-mediatedprotease formation was carried out by fresh isolation of the primaryclones and subsequently by isolation of the relevant insert-containingpUC18 vectors, retransformation and fresh screening (as above; secondaryplating). The transformants following from this likewise showed haloformation on skimmed milk medium and thus confirmed the location of aprotease gene on the DNA fragment cloned in each case.

Example 4 Sequence Analysis of a Proteolytically Active Clone (HP70Pa_(—)2)

From a protease-positive clone having the designation HP70 Pa_(—)2obtained according to Example 3, the plasmid DNA was isolated accordingto standard methods, the insert was prepared by means of Sac I/Hind IIIdigestion (see above) and sequenced according to standard methods. Here,first the primers specific for the vector and flanking the insertaccording to SEQ ID NO. 1 and 2 having the designations M13f and M13rwere used, followed by the “primer walking”, as is known from the priorart (R. J. Kaiser et al., “Specific primer-directed DNA sequencing usingautomated fluorescence detection”; Nucl. Acids Res., 17 (15) (1989), pp.6087-6102).

The sequencing of this clone afforded a region having an open readingframe whose DNA sequence is indicated in SEQ ID NO. 3. On account of itsorigin, it is noted as the organism “unknown” there and it isadditionally stated that this sequence is to be attributed to a DNAisolate. Furthermore, on account of the present data (in particular bymeans of sequence comparisons, see below) it has to be assumed that thenucleotide positions 1 to 96 code for the signal peptide and altogetherthe coding region extends from 1 to 1746. SEQ ID NO. 4 discloses theamino acid sequence derived herefrom, having the same data with respectto the origin.

As the nearest similar enzyme described, an extracellular serineprotease (E.C. 3.4.21.-) from Xanthomonas campestris pv. campestris(ATCC 33913) was found, which at GenBank (National Center forBiotechnology Information NCBI, National Institutes of Health, Bethesda,Md., USA) carries the accession number NP_(—)636242. The homology at theamino acid level determined by means of the computer program Vector NTI®Suite 7.0, obtainable from InforMax, Inc., Bethesda, USA, with thespecified default parameters is 75.0% identity to HP70. Further proteinsfound in this search, which at the amino acid level still appear themost similar, are compiled in Table 1 below.

TABLE 1 Nearest similar sequences to HP70 found at the amino acid levelAccession Identity Number Description (%) NP_636242 extracellularprotease from Xanthomonas 75.0 campestris pv. campestris, strain ATCC33913 NP_641280 extracellular protease from Xanthomonas 73.5 axonopodispv. citri strain 306 NP_641281 extracellular protease from Xanthomonas60.8 axonopodis pv. citri strain 306 NP_641282 extracellular proteasefrom Xanthomonas 60.8 axonopodis pv. citri strain 306 NP_636245extracellular protease from Xanthomonas 59.2 campestris pv. campestrisstrain ATCC 33913

At the DNA level, an identity of 74.4% results to the gene of theextracellular serine protease (E.C. 3.4.21.-) from Xanthomonascampestris pv. campestris (gene ID XCC0851).

Thus, the protease found is most highly probably likewise a serineprotease. A homology of 26.2% identity to the established B. lentusalkaline protease (WO 92/21760 A1) results at the amino acid level andan identity of 33.6% at the nucleic acid level.

The associated vector having the designation 70-pUC(AWB403) wasdeposited on 10.2.2003 at the Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH, Mascheroder Weg 1b, 38124 Brunswick and there carriesthe deposit number DSM 15977. The protease encoded hereby is designatedas HP70.

Example 5 Sequence Analysis of a Proteolytically Active Clone(HP53Pa_(—)2)

From a further protease-positive clone having the designationHP53Pa_(—)2 obtained according to Example 3, the insert was prepared andsequenced as in Example 4. The sequences obtained are shown in SEQ IDNO. 6 and 7. On account of its origin, it is noted there as organism“unknown” and it is additionally indicated that these sequences are tobe attributed to a DNA isolate. Furthermore, on account of the presentdata (in particular by means of sequence comparisons, see below) it hasto be assumed that the nucleotide positions 1 to 114 code for the signalpeptide and altogether the coding region extends from 1 to 1761.

It is in turn a subtilisin protease, which at the amino acid level has ahomology of 75.4% identity to the extracellular serine protease (E.C.3.4.21.-) from Xanthomonas campestris pv. campestris (ATCC 33913; seeabove) determined also in this case as the nearest similar. Furtherproteins found in this search which at the amino acid level still appearto be the most similar are compiled in table 2 below.

TABLE 2 Nearest similar sequences found to HP53 at the amino acid levelAccession Identity Number Description (%) NP_636242 extracellularprotease from Xanthomonas 75.4 campestris pv. campestris strain ATCC33913 NP_641280 extracellular protease from Xanthomonas 72.6 axonopodispv. citri strain 306 NP_641281 extracellular protease from Xanthomonas59.3 axonopodis pv. citri strain 306 NP_641282 extracellular proteasefrom Xanthomonas 59.8 axonopodis pv. citri strain 306 NP_636245extracellular protease from Xanthomonas 59.0 campestris pv. campestrisstrain ATCC 33913

At the DNA level, an identity of 75.0% results to the gene of theextracellular serine protease (E.C. 3.4.21.-) from Xanthomonascampestris pv. campestris.

At the amino acid level a homology of 25.9% identity and at the nucleicacid level an identity of 33.5% result to the established B. lentusalkaline protease (WO 92/21760 A1).

The associated vector having the designation 53-pUC(AWB403) wasdeposited on 10.2.2003 at the Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH, Mascheroder Weg 1b, 38124 Brunswick and there carriesthe deposit number DSM 15976. The protease encoded hereby is designatedas HP53.

Example 6 Production of C-Terminal Deletion Mutants of HP70 and HP53

HP70dc

C′-terminally, 332 bp were deleted from the protease of the clone havingthe designation HP70Pa_(—)2 obtained according to Example 3 anddescribed in Example 4. This amino acid sequence HP70_deltaC is shown inSEQ ID NO. 5. On account of its construction, it is noted there that itis a synthetic sequence, concretized with the detail “DNA isolate, DeltaC”. According to SEQ ID NO. 3 and 4, the section of positions 1 to 32can in turn be considered as a signal peptide.

For this, a 1413 by size DNA piece is generated from the DNA indicatedin SEQ ID NO. 3 using the oligonucleotides HP70f (SEQ ID NO. 10) andHP70r (SEQ ID NO. 11) as primers under standard PCR conditions. Afterprocessing the fragment obtained with the endonucleases EcoRI and BamHI,the fragment is cloned into the corresponding cleavage sites of the E.coli expression vector pUC18 and transformed in a suitable strain (forexample E. coli DH12S). From the transformants obtained, by blue/whiteselection the desired candidate can be identified, actively expressedand thus obtained in a sufficient amount for further investigations.

HP53dc

C′-terminally, 330 by were deleted from the protease of the clone havingthe designation HP53Pa 2 obtained according to Example 3 and describedin Example 5. This amino acid sequence HP53_deltaC is shown in SEQ IDNO. 8.

For this, a 1303 by size DNA piece is generated from the DNA indicatedin SEQ ID NO. 6 using the oligonucleotides HP53f (SEQ ID NO. 12) andHP53r (SEQ ID NO. 13) as primers under standard PCR conditions. Afterprocessing the fragment obtained using the endonucleases BlpI and BamHI,the fragment is cloned into the corresponding cleavage sites of the E.coli expression vector pUC18_HP70_dc obtained above in the first part ofthe example, which has been processed beforehand using the sameendonucleases.

After transformation in a suitable strain (for example E. coli DH12S),the desired candidate can be identified, actively expressed and thusobtained in an adequate amount for further investigations from thetransformants obtained by restriction analysis using SacII (pUC_HP53_dccontains such a cleavage site).

On account of this construction, it is noted in SEQ ID NO. 8 that it is,as in the case of SEQ ID NO. 5, a synthetic sequence, concretized withthe detail “DNA isolate, Delta C”. Furthermore, the section of positions1 to 32 according to SEQ ID NO. 5 was introduced here, such that thishas to be considered as a signal peptide of HP70.

Example 7 Quantitative Obtainment of the Proteases According to theInvention and Their Biochemical Characterization

The expression clones obtained according to Examples 3 to 6 were takenup in 100 ml of LB medium (10 g/l tryptone, 5 g/l, yeast extract, 10 g/lNaCl) and cultured in a 500 ml Erlenmeyer flask at 37° C. and withshaking at 200 rpm.

Subsequently, they were biochemically characterized. Here, theproteolytic activity was determined by means of an “MTP assay”, which isbased on a fluorescence-coupled casein substrate (BODIPY® FL Conjugate,Molecular Probes, Göttingen, Germany; Order No. #6638), to whichfluorophors (emitters) and dampers (quenchers) are coupled. In theintact substrate, fluorescence of the emitters is suppressed by thequenchers. On hydrolysis of the casein, the oligopeptides with thegroups coupled to them move away from one another and on correspondingexcitation fluorescence emission occurs, whose intensity is thus ameasure of the proteolysis.

For the activity determination, in each case 5 μl of a protease sampleaccording to example 5 are incubated in 100 mM Tris/HCl having thedesired pH and 4.5 μg/ml BODIPY® FL Conjugate I in a total volume of 100μl for 1 h at the temperature of interest. All measurements indicatedbelow were carried out in 96-well microtiter plates (Opaque® Plates,black; Corning BV Life Sciences, Schiphol-Rijk, Netherlands; Order No.#3915) with the aid of a FLUOstar® fluorescence measuring apparatus (BMGLab Technologies, Offenburg, Germany).

pH and Temperature Optimum

For the protease HP53 shown in SEQ ID NO. 7, the following biochemicalparameters result: pH optimum at 37° C.: 8.6 and temperature optimum atpH 8.6: 37° C.

Influence of Complexing Agents

The influence of complexing agents was investigated by addition of 1 mMEDTA at pH 8.6 in the assay indicated above, namely at 37° C. and 50° C.The measured value without addition of EDTA was set at 100%. Incontrast, the relative proteolytic activity at 50° C. was 27% and at 37°C. was 10%.

Stability Measurement

For measurement of the stability, the protease sample employed was firstpreincubated for 15 min at 50° C. in 50 mM NaHCO₃ buffer, pH 10.9 andthen the residual activity in the abovementioned assay was measured at37° C. and 50° C. at pH 8.6 in each case. Here, the activity of the sameextract without preincubation but otherwise identical treatment was ineach case set at 100%. In this way, a residual activity of 11% wasdetermined for 37° C. and of 13% for 50° C.

These are thus molecules which are relatively stable to high pHs, andthis even nearly independently of the temperature.

Example 8 Contribution of the Protease HP70 According to the Inventionto the Washing Performance at Relatively Low Temperature

For this example, standardized textiles provided with soilings wereemployed, which had been ordered from EidgenossischeMaterial-Prufungs-und-Versuchsanstalt, St. Gallen, Switzerland (EMPA).In this case, the following soilings and textiles were used: A(blood/milk/drawing ink on cotton), B (blood/milk/drawing ink on apolyester-cotton mixed fabric) and C (egg/soot on cotton).

Using this test material, various detergent recipes were investigatedlaunderometrically for their washing performance. For this, in each casea liquor ratio of 1:12 was set and washing was carried out for 30 min ata temperature of 40° C. The dosage was 5.9 g of the respectivecomposition per 1 of washing liquor. The water hardness was 16° Germanhardness.

The control detergent used was a detergent basis recipe of the followingcomposition (all data in percent by weight): 4% linearalkylbenzenesulfonate (sodium salt), 4% C₁₂-C₁₈ fatty alcohol sulfate(sodium salt), 5.5% C₁₂-C₁₈ fatty alcohol with 7EO, 1% sodium soap, 11%sodium carbonate, 2.5% amorphous sodium disilicate, 20% sodium perboratetetrahydrate, 5.5% TAED, 25% zeolite A, 4.5% polycarboxylate, 0.5%phosphonate, 2.5% foam inhibitor granules, 5% sodium sulfate, remainder:water, optical brightener, salts.

It was treated in parallel batches in each case activity-identicallywith the protease according to the invention and a control protease. Forthe control, the B. lentus alkaline protease F49 (WO 95/23221 A1;manufacturer: Biozym, Kundl, Austria) was used. This had a (determinableaccording to the method indicated in the description) specific activityof about 200 000 PE/g, whereby with 0.2% by weight an F49 concentrationof about 40,000 PE per 100 g of the composition and an activity of about2400 PE per 1 of washing liquor resulted. Recipes were additionallyprepared which, dispensing with a corresponding amount of salts, in eachcase contained 0.5%, that is the two and a half-fold amount of protease.The protease according to the invention was added to the same baserecipe in the same activity concentrations. In this regard, the % byweight values indicated for F49 in the table below are correct and applyfor HP70 by way of approximation.

After washing, the degree of whiteness of the washed textiles wasmeasured in comparison to that of barium sulfate, which had beenstandardized to 100%. The measurement was carried out on a DatacolorSF500-2 spectrometer at 460 nm (UV trap filter 3), 30 mm diaphragm,without gloss, light type D65, 10°, d/8°. The results obtained arecompiled as percent reflection, that is, as percentages in comparison tobarium sulfate together with the respective initial values in table 3below. The mean values from in each case three measurements areindicated. They allow an immediate conclusion on the contribution of theenzyme present to the washing performance of the composition used.

TABLE 3 Contribution of the protease HP70 according to the invention tothe washing performance at a temperature of 40° C. Base detergentcontaining A B C Initial value 14.1 12.8 28.9 Control without protease18.9 16.0 51.3 0.2% HP70 26.7 26.5 68.3 0.2% B. lentus alkaline proteaseF49 25.7 25.6 68.1 0.5% HP70 33.4 29.8 68.4 0.5% B. lentus alkalineprotease F49 30.0 32.2 70.2 Standard dev. 1.3 1.3 1.8

All three measuring series verify that the protease HP70 according tothe invention compared to protease-free detergents affords animprovement in the washing performance on protein-containing soilings.That is, it also displays a proteolytic activity in the presence ofdenaturing agents such as, for example, surfactants. The valuesdetermined for the B. lentus alkaline protease F49, a molecule optimizedfor this use area by means of point mutagenesis (compare WO 95/23221 A1)verify the correctness of the test procedure. In the measuring series Aand at the lower concentration in measuring series B, HP70 exceeded eventhe results for F49; the other values are comparable to those for F49.

Example 9 Contribution of the Protease HP53 According to the Inventionto the Washing Performance at Relatively Low Temperature

The previous test was repeated using the protease HP53 according to theinvention. The conditions were the same. However, the performance wasdetermined on the soiling D instead of on the soiling C (blood oncotton). The result is compiled in Table 4 below.

TABLE 4 Contribution of the protease HP53 according to the invention tothe washing performance at a temperature of 40° C. Base detergentcontaining A B C Initial value 13.5 13.9 18.8 Control without protease20.1 18.7 67.3 0.2% HP53 48.1 60.7 73.7 0.2% B. lentus alkaline proteaseF49 25.5 27.9 69.7 0.5% HP53 42.9 53.5 69.0 0.5% B. lentus alkalineprotease F49 32.6 41.1 73.8 Standard dev. 4.5 1.7 2.4

These measuring series verify that the protease HP53 according to theinvention also affords an improvement of the washing performance onprotein-containing soilings compared to protease-free detergents. Thatis, it also displays a proteolytic activity in the presence ofdenaturing agents such as, for example, surfactants or bleach. Thevalues determined are comparable at least to those for the B. lentusalkaline protease F49, even distinctly superior in the measuring seriesA and B.

Example 10 Contribution of the Protease HP53dc According to theInvention to the Washing Performance at Relatively Low Temperature

As in the preceding two Examples, the variant HP53dc was alsoinvestigated with respect to its performance contribution with regard tothe soilings A, B and C. The conditions were again the same. The resultis compiled in Table 5 below.

TABLE 5 Contribution of the protease HP53dc according to the inventionto the washing performance at a temperature of 40° C. Base detergentcontaining A B C Initial value 14.6 13.2 28.1 Control without protease19.6 16.2 54.0 0.2% HP53dc 42.8 41.2 71.2 0.2% B. lentus alkalineprotease F49 26.8 31.2 70.0 0.5% HP53dc 48.6 50.0 70.8 0.5% B. lentusalkaline protease F49 29.8 40.7 70.9 Standard dev. 1.6 1.9 2.5

These measuring series verify that the protease HP53dc according to theinvention also affords an improvement of the washing performance onprotein-containing soilings compared to protease-free detergents. Thatis, it displays a proteolytic activity even in the presence ofdenaturing agents such as, for example, surfactants or bleach. Thevalues determined in the measuring series A and B are clearly superiorto those for the B. lentus alkaline protease F49 and in series C atleast comparable.

Example 11 Contribution of the Protease HP53dc According to theInvention to the Washing Performance at Relatively High Temperature

The preceding test was repeated with HP53 on the two soilings A and B ata washing temperature of 60° C. under otherwise identical conditions.The result is compiled in table 6 below.

TABLE 6 Contribution of the protease HP53dc according to the inventionto the washing performance at a temperature of 60° C. Base detergentcontaining A B Initial value 14.6 13.2 Control without protease 20.516.7 0.2% HP53dc 36.3 36.9 0.2% B. lentus alkaline protease F49 25.632.4 0.5% HP53dc 47.7 48.0 0.5% B. lentus alkaline protease F49 31.443.5 Standard dev. 1.7 1.3

Even at the temperature of 60° C., in the measuring series A and B thesuperiority of the protease HP53dc according to the invention to the B.lentus alkaline protease F49 is seen. Fortunately, the protease HP53dcaccording to the invention at 60° C. is not noticeably denatured, suchthat it is in particular suitable as a detergent protease.

1. An isolated protein comprising an amino acid sequence which is atleast 95% identical to SEQ ID NO: 7, wherein said sequence has alkalineprotease activity.
 2. The isolated protein of claim 1, wherein thesequence is at least 95% identical to the amino acid positions 39 to 586of SEQ ID NO.
 7. 3. The isolated protein of claim 1, said sequence beingobtained by insertion mutagenesis or by substitution mutagenesis.
 4. Theisolated protein of claim 1 which is additionally stabilized.
 5. Theisolated protein of claim 1, which is additionally derivatized.
 6. Acomposition comprising the isolated protein of claim
 1. 7. Thecomposition of claim 6, which is a detergent or cleaner.
 8. Thecomposition of claim 7 wherein the isolated protein is present in anamount from about 2 μg to 20 mg per gram of the composition.
 9. Thecomposition of claim 8, wherein the isolated protein is present in anamount from about 50 μg to 10 mg per gram of the composition.
 10. Thecomposition of claim 6, further comprising one or more enzymes, said oneor more enzymes selected from the group consisting of proteases,amylases, cellulases, hemicellulases, oxidoreductases and lipases. 11.The composition of claim 7, which is a composition for the washing orcleaning of textile raw materials or fibers or textiles containing woolor silk.
 12. An isolated protein, wherein the isolated protein comprisesthe amino acid sequence according to positions 39 to 586 of SEQ ID NO:7.
 13. An isolated fusion protein comprising the isolated protein ofclaim 1 which is further modified by fusion with at least one otherprotein.